KR20020057945A - Method of correlating sequence function by transfecting a nucleic acid sequence of a donor organism into a plant host in an anti-sense or positive sense orientation - Google Patents

Abstract

The present invention relates to a method of correlating the function of a nucleic acid sequence derived from a host organism by transient expression of the nucleic acid sequence in the antisense or positive sense direction in a host plant.

Description

METHOD OF CORRELATING SEQUENCE FUNCTION BY TRANSFECTING A NUCLEIC ACID SEQUENCE OF A DONOR ORGANISM INTO A PLANT HOST IN AN ANTI -SENSE OR POSITIVE SENSE ORIENTATION}

There is great interest in starting human and non-human genome projects. The human genome is 2.8 million to 3.5 million base pairs, of which about 3% is composed of genes. In June 2000, the human genome project and biotech company Celera Genomics announced the completion of a rough draft of the human genome (http://www.ncbi.nlm.nih.gov). However, this information will only indicate the reference sequence of the human genome. What remains is in the determination of sequence function, which is important in the study, diagnosis and treatment of human disease.

The sequence of the mouse genome is also determined. Genbank provides about 1.2% of the mouse genome of 3 billion bases (http://www.informatics.jax.org) and the rough draft of the mouse genome is available until 2003, and the genome will be completed by 2005 It is expected. In addition, the Drosophila genome project has recently been completed (http://www.fruitfly.org).

Valuable and basic crops, including corn, soybeans and rice, are also targets for the genome project, as the information obtained thereby can prove to be very beneficial in increasing global food production and improving the quality and value of crops . The US Congress is considering starting the corn genome project. By helping to solve the genetics concealed in the corn genome, the project will help understand and cure common diseases of grain crops. It could also provide a great boost to efforts to manipulate plants to improve grain yields and to resist drought, disease, salt, and other extreme environmental conditions. Such progress is important to the world population, which is expected to double by 2050. At present, there are four species of wheat, rice, maize, and potato that provide 60% of all human food, and strategies to increase the productivity of these plants include rapid detection of the presence of traits in these plants, It depends on the function of the sequence.

One strategy proposed to help such efforts is to build a database of expressed sequence tags (ESTs) that can be used to identify expressed genes. Accumulation and analysis of expressed sequence tags (ESTs) have become an important part of genome research. EST data can be used to identify gene products and thereby speed up gene cloning. Various sequence databases have been established in an effort to store and correlate vast amounts of sequence information generated by ongoing sequencing efforts. Some have presented 500,000 ESTs for corn and 100,000 ESTs for rice, wheat, oats, barley, and sorghum, respectively. Efforts to determine the genomic sequence of plant species will undoubtedly depend on such a computer database to share sequence data as sequences are generated. Arabidopsis thaliana has shown that a very large set of ESTs have already been generated in this organism and this sequence is more than 50% of the expected Arabidopsis gene, have.

The potential use of such generated sequence information is enormous if gene function can be determined. It will be possible to manipulate commercial seeds for agricultural use to transfer a certain number of desirable traits to food and fiber crops to increase agricultural production and global food supply. Research and development of commercial seeds has focused primarily on traditional plant breeding, but biotechnology is increasingly interested in plant characteristics. The knowledge of the genome involved in both terminal and dicotyledons and the function of the genes involved is essential to realizing positive effects from such techniques.

The impact of genomic research on seeds is potentially great. For example, a gene profile in cotton can help to understand the type of gene that is preferentially expressed in fibroblasts. Genes or promoters derived from these genes may be important in the genetic engineering of cotton fibers for increased strength or " built-in " fiber pigments. In plant breeding, the gene profile associated with the analysis of physiological characteristics will enable identification of the expected marker, which is of considerable importance in the marker-assisted breeding program. Finding the DNA sequence of a particular crop for a gene that is important to harvest, quality, health, appearance, color, taste, etc. is clearly an important attempt to improve the crop.

Studies have been conducted in the development of suitable vectors expressing foreign DNA and RNA in plant and animal hosts. [Ahlquist, U.S. Patent Nos. 4,885,248 and 5,173,410] describe a preliminary study devising a delivery vector that may be useful for delivery of foreign genetic material to a host plant for expression purposes. Additional aspects of hybrid RNA viruses and RNA transformation vectors are described in [Ahlquist et al., U.S. Pat. Nos. 5,466,788, 5,602,242, 5,627,060 and 5,500,360]. [Donson et al., U.S. Pat. Nos. 5,316,931, 5,589,367 and 5,866,785] first demonstrate plant virus vectors suitable for systemic expression of foreign genetic material in plants. Donson et al. Describe plant virus vectors with heterologous subgenomic promoters for systemic expression of foreign genes. [Carrington et al., U.S. Patent 5,491,076] describe a specific potyvirus vector also useful for expression of foreign genes in plants. The expression vector described by Carrington et al. Is characterized by the unique ability of viral polyprotein proteases to cleave heterologous proteins from viral multiproteins. This includes potiviruses such as tobacco rust viruses. Additional suitable vectors are described in U.S. Patent No. 5,811,653 and U.S. Patent Application Serial No. 08 / 324,003. Condreay et al. ( Proc Natl Acad Sci USA 96 : 127-132) disclose efficient delivery and expression of genes in cell types of human, primate, and rodent origin using Baculovirus. Price et al . ( Proc. Natl. Acad. Sci. USA 93 : 9465-9570 (1996)) disclose infecting insect, plant and mammalian cells with Nodavirus.

Construction of plant RNA viruses for introducing and expressing non-viral foreign genes into plants is also described in Brisson et al., Methods in Enzymology 118 : 659 (1986), Guzman et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, . 172-189 (1988), Dawson et al., Virology 172 : 285-292 (1989), Takamatsu et al., EMBO J. 6 : 307-311 (1987), French et al., Science 231 : 1294-1297 (1986), and Takamatsu et al., FEBS Letters 269 : 73-76 (1990). However, these viral vectors have shown that systemic transformation in plants and the expression of non-viral foreign genes in the vast majority of plant cells within the whole plant can not be achieved. Moreover, many of these viral vectors have proven not to be stable to the maintenance of nonviral foreign genes. However, the viral vectors described in [Donson et al., U.S. Pat. Nos. 5,316,931, 5,589,367, and 5,866,785, Turpen, U.S. Pat. No. 5,811,653, Carrington et al., U.S. Pat. No. 5,491,076, and co-pending U.S. patent application Serial No. 08 / , It has been demonstrated that plant cells can be infected with foreign genetic material, systemically transferred into plants, and localized or systemically expressed non-viral foreign genes contained within plant cells. Morsy et al. ( Proc Natl Acad Sci USA 95: 7866-7871 (1998)) have developed adjuvant-dependent adenovirus vectors that have an insertion capacity of 37 kb or less and can be easily propagated.

With the recent arrival of cloning technology, genes can be selectively lost. One method is to selectively bind to the RNA message of the target gene to create an antisense RNA or DNA molecule that interferes with the precise molecular mechanism by which the gene is expressed as a protein. Antisense technology that deactivates certain genes provides a different approach to classical genetic approaches. Classical genetics typically studies random mutations of all genes in organisms and selects mutations that are specific to a particular feature. The antisense approach begins with the cloned gene of interest and manipulates it to elicit information about its function.

Expression of a virus-like positive sense or antisense RNA in a transgenic plant provides for increased or decreased expression of an endogenous gene. In most cases, the introduction of the transgene and its subsequent expression increases the steady-state level of certain gene products (using positive sense RNA) or decreases (using antisense RNA) ( Curr. Opin. Cell Biol . 7 : 399-405 (1995)). There is also evidence that inhibition of the innate gene occurs in transgenic plants containing sense RNA (Van der Krol et al., Plant Cell 2 (4): 291-299 (1990), Napoli et al., Plant Cell 2: 279-289 1990) and Fray et al., Plant Mol. Biol. 22: 589-602 (1993)).

Post transcriptional gene silencing (PTGS) in transgenic plants is an indication of a mechanism that inhibits RNA accumulation in a sequence-specific manner. There are three models that illustrate the mechanism of PTGS: antisense RNA produced in response to the direct transcription of antisense RNA from transgene, overexpression of transgene, or antisense RNA produced in response to the production of a false sense RNA product of transgene (Baulcombe , Plant Mol Biol., 32: 79-88 (1996)). Post-transcriptional gene silencing machinery is typified by highly specific degradation of both transgene mRNA and target RNA, including either the same or a complementary base sequence. When the silencing transgene is the same sense as the target-bearing gene or the viral genomic RNA, the RNA-dependent RNA polymerase encoded by the plant produces a complementary strand from the transgene mRNA and a small cRNA enables the degradation of the target RNA . It has been proposed that antisense RNA and hypothetical cRNA act by hybridizing with the target RNA to render the hybrid a substrate for double-stranded RNA degrading enzymes or to stop translation of the target RNA (Baulcombe, Plant Mol. Biol. 32: 79- 88 (1996)). It is also proposed that said down-regulation or " simultaneous inhibition " by sense RNA can be due to the production of antisense RNA by over-all transcription from an end promoter located on the opposite strand of chromosomal DNA (Grierson et al., Trends Biotechnol. 122-123 (1993)).

Waterhouse et al . ( Proc. Natl. Acad. Sci. USA 10: 13959-64 (1998)) produced transgenic tobacco plants containing sense or antisense constructs. The Pro [s] and Pro [a / s] constructs contained PVY nuclear inclusion Pro ORF in the sense and antisense orientation, respectively. Pro [s] - The stop construct contained the PVY Pro ORF in the sense direction, but had a stop codon three codons downstream from the start codon. Waterhouse et al. Showed that when the construct gene was transfected into a plant, the plant immunized against the transgene-derived virus form. Smith et al. (Plant Cell, 6: 1441-1453 (1994)) produced a tobacco transgenic plant containing the potato virus Y (PVY) envelope protein (CP) open reading frame, MRNAs that could not be translated by introduction were produced. The expression of non-transcribable sense RNA is inversely related to the viral resistance of transgenic plants. Kumagai et al . ( Proc. Natl. Acad. Sci. USA 92: 1679 (1995)) showed that gene expression in transfected Nicotiana bentamiana leads to the expression of lycopersicon esculentum phytoene unsaturation in a positive sense or antisense orientation Lt; RTI ID = 0.0 > cytoplasmic < / RTI > by viral delivery of RNA of known sequence derived from the encoding cDNA.

Antisense and positive sense technologies can be used to develop functional genomic screening of donor organisms from prokaryotes (Monera), protista, fungi, plants (Plantae) or animals (Animalia). The present invention provides a method for determining the function and sequence of a nucleic acid of a donor organism by detecting the presence of a property in the host plant and expressing the nucleic acid sequence in the host plant. GTP-binding proteins exemplify the invention. In eukaryotic cells, GTP-binding proteins function in a variety of cellular processes, including signal transduction, cytoskeletal tissue, and protein transport. A low molecular weight (20-25 kilodalton) GTP-binding protein binds to ras and its close species (e.g. Ran), rho and its close species, the rab family, and the ADP-ribosomal factor (ARF) family . Heterogeneous trimer and monomeric GTP-binding proteins that may be involved in secretion and intracellular transport are subdivided into two structural classes: rab and the ARF family. Ran, a small soluble GTP-binding protein, has been shown to be essential for protein nuclear migration and is thought to be involved in regulating cell cycle progression in mammalian and yeast cells. CDNA encoding the GTP-binding protein has been isolated from a variety of plants including rice, barley, corn, tobacco, and A. thaliana. For example, Verwoert et al. (Plant Molecular Biol. 27: 629-633 (1995)) encode the GTP-binding protein of the ARF family by direct genetic selection in E. coli fabD mutants using a maize cDNA expression library We report the isolation of Zea mays cDNA clones. Regad et al. (FEBS 2: 133-136 (1993)) randomly selected cDNA clones and sequenced to isolate cDNA clones encoding ARF from cDNA libraries of Arabidopsis thaliana cultured cells. Dallmann et al. (Plant Molecular Biol. 19: 847-857 (1992)) used a PCR approach to isolate two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries. Dallmann et al. Used leaf cDNA as a template and used them as primers in PCR amplification using degenerate oligonucleotides corresponding to highly conserved motifs found in the ras super family. Haizel et al. (Plant J., 11: 93-103 (1997)) isolated cDNA and genomic clones encoding Ran-like small GTP-binding proteins from Arabidopsis cDNA and genomic libraries using full length tobacco Nt Ran1 cDNA as a probe . The present invention provides advantages over this method in determining the nucleic acid sequence encoding the GTP-binding protein in that it only determines the sequence of the functioning clone and does not randomly determine the sequence of the clone. Lt; RTI ID = 0.0 > cDNA < / RTI > hybridizing thereto.

The present invention claims priority from U.S. Patent Application Serial Nos. 09 / 359,301, 09 / 359,305, 09 / 359,297, and 09 / 359,300 filed on July 21, 1999.

The present invention relates generally to the field of molecular biology and genetics. Specifically, the present invention relates to a method of correlating the function of a nucleic acid sequence derived from a host organism by transient expression of the nucleic acid sequence in the antisense or positive sense direction in the host plant.

Figure 1 shows the plasmid pBS # 735.

Figure 2 shows plasmid pBS # 740.

Figure 3 shows the plasmid TTU51A QSEO # 3.

Figure 4 shows the plasmid TTO1A / Ca CCS +.

Figure 5 shows the plasmid TTOl / PSY +.

Figure 6 shows the plasmid TTOl / PDS +.

Figure 7 shows a terminal lobe viral vector.

Figure 8 shows the plasmid TTU51 CTP CrtB.

Figure 9 shows the plasmid pBS 740 AT # 2441 (ATCC number: PTA-332).

Figure 10 shows the nucleic acid sequence of 740 AT # 2441.

Figure 11 shows a nucleic acid sequence comparison of 740 AT # 2441 and AF017991.

Figure 12 shows nucleic acid sequence comparisons of 740 AT # 2441 and L16787.

Figure 13 shows amino acid sequence comparison of 740 AT # 2441 and RAN-B1 GTP binding protein.

14 shows plasmid pBS 740 AT # 120 (ATCC number: PTA-325).

Figure 15 shows a nucleic acid sequence comparison of Arabidopsis thaliana 740 AT # 120 and Arabidopsis thaliana est AA042085.

Figure 16 shows the nucleic acid sequence alignment of 740 AT # 120 to rice D17760 (Oryza sativa) ADP-ribosylation factor.

Figure 17 shows the nucleic acid sequence alignment of 740 AT # 120 to the human ADP-ribosylation factor P16587.

Figure 18 shows the nucleic acid sequence alignment of the humanized sequence 740 AT # 120 H to human ADP-ribosylation factor M33384.

Figure 19 shows the plasmid KS + Nb ARF # 3 (ATCC number: PTA-324).

Figure 20 shows a nucleic acid sequence comparison of Arabidopsis thaliana 740 AT # 120 and Nicotiana botanicumina KS + Nb ARF # 3.

Figure 21 shows the tobacco rattle virus gene silencing vector.

Figure 22 shows the plasmid pBS # 740 AT # 88 (ATCC number: PTA-331).

Figure 23 shows the sequence of 740 AT # 88.

Figure 24 shows a nucleic acid sequence comparison of AT # 88 and Brassica rapa L35812.

Figure 25 shows a nucleic acid sequence comparison of AT # 88 and Octopus Rhodopsin X07797.

Figure 26 shows nucleic acid sequence comparisons of AT # 88 and Octopus Rhodopsin P31356.

Figure 27 shows the plasmid pBS # 377 (ATCC number: PTA-334).

Figure 28 shows the nucleic acid sequence of 740 AT # 377.

29 shows the plasmid pBS # 2483 (ATCC number: PTA-329).

Figure 30 shows the nucleic acid sequence of 740 AT # 2483.

Figure 31 shows the plasmid pBS 740 AT # 909 (ATCC number: PTA-330).

32 shows a nucleic acid sequence comparison of AT # 909 and the ribosomal protein L19 of the breast cancer cell line.

33 shows a nucleic acid sequence comparison of AT # 909 and L19 P14118 60S ribosomal protein L19.

Figure 34 shows the plasmid pBS AT # 855 (ATCC number: PTA-325).

Figure 35 shows nucleic acid sequence comparisons of AT # 855 and HAT7 homeobox protein ORF.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in principle to: (1) introducing into a viral vector a library of sequence inserts derived by a host organism in a positive or antisense orientation; (2) expressing each insert in a host plant, and (3) detecting phenotypic or biochemical changes in the host plant as a result of expression. Host plants can be terminal or dicot plants, plant tissues or plant cells. Donor organisms include species derived from prokaryotes, protists, fungi, plants or animals, such as humans, mice, fruit flies, and the like. If the donor organism is also a plant, the donor plant and the host plant typically belong to different genus, tree, tree, river, moon, or gate. The function of a sequence insert in a library is typically unknown. The number of sequence inserts in the library is typically greater than about 10, 15, 20, 50, 100, 200, 500, 1000, 5000, or 15,000, The length of each insert is typically longer than about 50, 100, 200, or 500 base pairs.

More particularly, the invention relates to a method of altering the phenotype or biochemistry of a host plant, a method of determining phenotypic or biochemical changes in a host plant, and a method of determining the presence of a property in a host plant. The method comprises transiently expressing a nucleic acid sequence of a donor organism in a host plant in the antisense or positive sense direction, identifying the change in the host plant, and correlating the phenotype or biochemical change with sequence expression. The nucleic acid sequence need not be isolated, identified, or characterized prior to transfection into the host organism.

The present invention also relates to a method of transiently expressing a nucleic acid sequence library in a host organism, determining a phenotypic or biochemical change in the host plant, identifying characteristics associated with the change, identifying the donor gene associated with the trait, Identifying a homologous host gene and annotating the sequence as a phenotype or function associated therewith.

The present invention also relates to a method for determining the function of a nucleic acid sequence, including a gene in a donor organism, by transfecting the nucleic acid sequence in a host plant in a manner to effect phenotypic or biochemical changes in the host plant. In one embodiment, the recombinant viral nucleic acid is prepared to include a donor nucleic acid insert. The recombinant viral nucleic acid infects host plants and produces antisense or positive sense RNAs in the cytoplasm that result in reduced or increased expression of cellular genes that are internal to the host organism. Once the presence of the property is confirmed by phenotypic or biochemical changes, the function of the nucleic acid is determined. The nucleic acid sequence of the cDNA clone or nucleic acid insert in the vector is then determined. The nucleic acid sequence is determined by standard sequence analysis.

One aspect of the present invention is a method for identifying and determining a nucleic acid sequence in a donor organism wherein the function of the sequence is to introduce the nucleic acid into the host plant via a viral nucleic acid suitable for expressing the nucleic acid in the transfected plant, The function of the inherent gene is lost. This method utilizes the principle of post-transcriptional gene silencing of an inherent host genetic animal, such as antisense RNA, or positive sense RNA. In particular, this silencing function is useful for silencing multigene families in donor organisms. In addition, overexpression of positive sense RNA, which results in overproduction of the protein, can lead to phenotypic or biochemical changes in the host.

Another aspect of the present invention is to find a gene in a donor organism having the same function as a gene in a host plant. The method begins by constructing a pool of cDNA libraries, or genomic DNA libraries, or RNAs of donor organisms from, for example, tissues or cells of a human, mouse, or fruit fly. The recombinant viral nucleic acid containing the nucleic acid insert derived from the library is then prepared and used to infect the host plant. Infected host plants are examined for phenotypic or biochemical changes. Recombinant viral nucleic acids that result in phenotypic or biochemical changes in the host plant are identified and the sequence of the nucleic acid insert is determined by standard methods. In a donor organism, such a nucleic acid sequence may have substantial sequence homology with the sequence in the host plant , For example, the nucleic acid sequence is conserved between the donor and the host plant. Once the nucleic acid sequence is determined, it can be labeled and used as a probe to isolate the full-length cDNA from the donor organism. The present invention provides a rapid means of identifying the function and sequence of a donor bio-nucleic acid; Information that expands so rapidly can then be used in the field of genomics.

Another aspect of the invention relates to a method for increasing the yield of grain crops. The method involves transiently expressing a nucleic acid sequence of a donor plant in the antisense or positive sense direction in a grain crop, which results in inhibited growth and increased seed production of the grain crop. A preferred method involves the step of cloning a nucleic acid sequence into a plant virus vector and infecting the crop with a recombinant viral nucleic acid containing said nucleic acid sequence.

Another aspect of the invention relates to a method of producing human proteins in host plants. After isolating and identifying nucleic acids of similar function from human and host plants, the amino acid sequences derived from the DNA are compared. The plant nucleic acid sequence is changed to encode the same amino acid sequence as the human protein. The nucleic acid sequence may be changed according to any conventional method such as site-directed mutagenesis or polymerase-based DNA synthesis.

Another aspect of the invention is to find genes with the same function in different plants. The method starts with the first plant cDNA, genomic DNA library, or RNA pool. The recombinant viral nucleic acid containing the nucleic acid insert derived from the library is then prepared and used to infect different host plants. Infected host plants are examined for phenotypic or biochemical changes. Identify recombinant viral nucleic acids that cause phenotypic or biochemical changes in host plants and determine the sequence of nucleic acid inserts by standard methods. Such a nucleic acid sequence in the first plant has substantial sequence homology with the sequence of the host plant: the nucleic acid sequence is conserved between the two plants. The present invention provides a rapid means of revealing the function and sequence of a nucleic acid of interest; Such rapidly expanding information can then be used in the field of genomics.

I. Introduction of a library of sequence inserts from donor organisms into plant virus vectors

Construction of the virus expression vector can be carried out by various methods known in the art. In a preferred embodiment of the invention, the viral vector is derived from an RNA plant virus. In particular, various plant viruses such as Bromoviridae, Bunyaviridae, Comoviridae, Geminiviridae, Potyviridae, and Tombusviridae can be used . In plant viruses, it is particularly preferred to carry out the method of the present invention in the presence of at least one compound selected from the group consisting of an alphavirus, an ilavirus, a bromovirus, a cucumovirus, a tospovirus, a colovirus, a collimovirus, a closterovirus, a comovirus, A virus selected from the group consisting of a virus, a virus, a virus, a virus, a virus, a virus, a virus, a virus, a rheumatoid, A variety of viruses such as thymovirus, Umbra virus, etc. may be suitable for the present invention.

Within the genus of plant viruses, many species are particularly desirable. They are particularly useful for the treatment of Alfalfa mosaic virus, cigarette striped virus, brome mosaic virus, beetle stain virus, Eastern blotch virus, cucumber mosaic virus, tomato spotted wilt virus, carnation latent virus, cauliflower mosaic virus, beet yellow virus, eastern mosaic Virus, cigarette round pattern virus, carnation round pattern virus, soil infection wheat mosaic virus, tomato gold mosaic virus, cassava latent virus, barley stripe mosaic virus, barley yellow atrophy virus, tobacco necrotic virus, tobacco corrosion virus, potato virus X, potato Virus Y, rice necrosis virus, taro mosaic virus, barley yellow mosaic virus, rice seed stunt virus, southern soybean mosaic virus, tobacco mosaic virus, rib Green mosaic virus, cucumber green stain mosaic virus, watermelon line, oat mosaic virus, tobacco rattle virus, carnation stain virus, tomato bush stunt virus, radish yellow mosaic virus, carrot stain virus. In addition, RNA satellite viruses such as tobacco necrosis can also be used.

The proposed plant viruses may contain DNA or RNA, which may be single or double stranded. One example of a plant virus containing double-stranded DNA includes, but is not limited to, collimovirus such as cauliflower mosaic virus (CaMV). Representative plant viruses containing single stranded DNA are the cassava latent virus, the soybean gold mosaic virus (BGMV), and the yellowish striped mosaic virus. Rice atrophy virus and wound tumor virus are examples of double-stranded RNA plant viruses. Single strand RNA plant viruses include tobacco mosaic virus (TMV), radish yellow mosaic virus (TYMV), rice necrosis virus (RNV), and bromine mosaic virus (BMV). Single-stranded RNA viruses can further be divided into positive sense (or positive strand), negative sense (or negative strand), or both sense viruses. The genomic RNA of the positive sense RNA virus is a sentence sense, which makes the exposed RNA infectious. Many plant viruses belong to the family of positive-sense RNA viruses. They include, for example, TMV, BMV, and others. RNA plant viruses typically contain replication enzyme / polymerase proteins that are essential for viral replication and mRNA synthesis, coat proteins that provide a protective shell against the extracellular pathway, and other proteins necessary for self-assembly of intercellular transport, And encodes several common proteins. For general information on plant viruses, see Matthews, Plant Virology, 3rd ed., Academic Press, San Diego (1991).

Selected groups of suitable plant viruses are identified below. However, the present invention should not be construed as limited to the use of such specific viruses, rather, the method of the present invention is believed to include at least all plant viruses. Should not be construed as limiting, rather, the present invention is believed to include all suitable viruses. Some suitable viruses are identified below.

Tobamovirus group

Tobacco mosaic virus (TMV) is of particular interest to the present invention due to its ability to express genes at high levels in plants. TMV is a member of the tobacco virus. TMV virion is a tubular filament and contains a coat protein subunit arranged in a single first strand with single strand RNA inserted between turns of the strand. TMV infects not only tobacco but also other plants. TMV virion is a 300 nm × 18 nm tube with a cavity of 4 nm in diameter and consists of 2140 units of a single structural protein spirally wound around a single RNA molecule. The genome is 6395 bases of positive sense RNA. The 5 'end is capped and the 3' end has a structure similar to a tRNA that specifically accepts a series of pseudoknot and histidine. Genomic RNA functions as an mRNA for the production of proteins involved in viral replication: a 126 kDa protein starting at 68 nucleotides from the 5 'terminus, and an 183 kDa protein synthesized by going through an arm termination codon at about 10% . Only 183 kDa and 126 kDa viral proteins are required as trans for TMV replication (Ogawa et al., Virology 185: 580-584 (1991)). Additional proteins are translated from subgenomic size mRNA produced during replication (Dawson, Adv. Virus Res., 38: 307-342 (1990)). 30 kDa protein is required for intercellular translocation; The 17.5 kDa capsid protein is a single viral structural protein. The expected function of the 54 kDa protein is unknown.

TMV assembly apparently occurs in the cytoplasm of plant cells, although transcripts of ctDNA have been detected in purified TMV virions suggesting that some TMV assembly may occur in chloroplasts. The onset of TMV assembly is caused by interactions between annular aggregates of envelope proteins ("discs") (each disk consists of two layers of 17 subunits) and a unique internal nucleation site in RNA; The hairpin region is at about 900 nucleotides from the 3 'end in the common system of TMV. Any RNA can be packaged as virion, including subgenomic RNA including this site. The disk apparently takes the form of a spiral when interacting with RNA, and assembly (growth) then proceeds in both directions (but progresses much more rapidly in the 3 'to 5' direction from the nucleation site).

Another member of the tobacco virus, the cucumber green stain mosaic virus watermelon line (CGMMV-W) is associated with the cucumber virus (Nozu et al., Virology 45: 577 (1971)). The coat protein of CGMMV-W interacts with both RNAs of TMV and CGMMV to assemble viral particles in vitro (Kurisu et al., Virology 70: 214 (1976)).

Several lines of the tovamo virus group are classified into two subgroups based on the assembly origin position. Subgroup I, including bone statistics, OM, and tomato lines, has about 800-1000 nucleotides from the 3 'end of the RNA genome, and an assembly origin outside the envelope protein sisterone (Lebeurier et al., Proc. Natl. Acad. 74: 149 (1977); and Fukuda et al., Virology 101: 493 (1980)). Subgroup II, including CGMMV-W and the consier system (Cc), has about 300-500 nucleotides from the 3 'end of the RNA genome and an assembly origin within the coat protein sisteron. The coat protein cistron of CGMMV-W is located at nucleotides 176-661 from the 3 'end. The 3 'unencrypted region is 175 nucleotides in length. The assembly origin is located within the coat protein sistron (Meshi et al., Virology 127: 54 (1983)).

Bromine mosaic virus group

Bromine mosaic virus (BMV) is a member of a family of plant viruses containing three molecules, single strand, RNA, commonly referred to as a virus. Each member of the viral virus infects a narrow range of plants. The mechanical propagation of the Bromovirus occurs easily, and some members are transferred by the beetle. In addition to BV, other Bromo viruses include vesicular staining virus and eastern yellow staining virus.

Typically, the Bromovirus virion is a dodecahedron with a diameter of about 26 microns, containing a single type of coat protein. Bromovirus genomes have linear, positive sense, single strand RNA molecules, and envelope protein mRNA is also capped. Each of the RNAs has a capped 5 ' end and a tRNA-like structure at the 3 ' end (accepting tyrosine). Virus assembly occurs in the cytoplasm. The complete nucleic acid sequence of BMV is described in Ahlquist et al., J. Mol. Biol. 153: 23 (1981).

Rice necrosis virus

Rice necrosis virus is a member of potato virus Y group or potivirus. Rice necrosis virions are curved filaments containing one type of coat protein (molecular weight of about 32,000 to about 36,000) and one molecule of linear positive sense single strand RNA. Rice necrosis virus is transmitted by Polymyxa oraminis (an eukaryotic parasite found in plants, birds and molds).

Gemini virus

Gemini viruses are a group of plant viruses that contain small, single stranded DNA with a distinctive form of virion. Each virion consists of a pair of equiaxed particles (incomplete chopped dodecahedra) composed of a single type of protein (molecular weight about 2.7-3.4 × 10 ⁴ ). Each gemini virus virion contains one molecule of circular, positive sense, single stranded DNA. In some gemini viruses (cassava latent virus and soybean gold mosaic virus), the genome appears to be a bimodil containing two single stranded DNA molecules.

Forty virus

Forty virus is a group of plant viruses that produce multiproteins. A particularly preferred potty virus is tobacco corrosion virus (TEV). TEV is a well-characterized potty virus and contains a 9.5 kb positive strand RNA genome that encodes a single large polyprotein that is cleaved by three virus-specific proteolytic enzymes. Nuclear-Involved Protein The "a" protein hydrolytic enzyme is involved in the maturation of several replication-related proteins and capsid proteins. Both the adjunct component-proteinase (HC-Pro) and the 35 kDa protein hydrolase catalyze cleavage at their respective C-termini. In each of these proteins, the proteolytic domain is located near the C-terminus. The 35 kDa protein hydrolase and HC-Pro are derived from the N-terminal region of the TEV polyprotein.

Hordievirus group

Hordei viruses are a group of plant viruses containing a single strand, positive sense RNA, with three or four partial genomes. The Hordei virus has a rigid vaginal virion and the Barley Stripe Mosaic Virus (BSMV) is a member of that type. BSMV infects a number of terminal and dicotyledon species including barley, oats, wheat, corn, rice, spinach, and Nicotiana bentamiana. Local damage hosts include Chenopodium amaranticolor, and Nicotiana tabacum ccv. Includes Samsun. BSMV is not propagated by the vector but propagates mechanically and in some hosts such as barley, through the pollen and seeds as well.

Most lines of BSMV have three genomic RNAs referred to as alpha (alpha), beta (beta), and gamma (gamma). At least one line, the Argentine mild (AM) line, inherently has the fourth genomic RNA, a deletion mutant of gRNA. All genomic RNAs are capped at the 5 'end and have a tRNA-like structure at the 3' end. Virus replication and assembly occurs in the cytoplasm. An infectious cDNA clone is available (Petty et al., Virology et al., (Reviewed by Jackson et al., Annual Review of Phytopathology, 27: 95-121 (1989)) and the complete nucleic acid sequence of several strains of BSMV has been identified and identified 171: 342-349 (1989)).

The choice of the genetic framework for the viral vectors of the present invention may depend on the host plant used. Host plants can be terminal or dicot plants, plant tissues, or plant cells. Typically, plants of commercial interest, such as edible crops, seed crops, oil crops, ornamental crops and forestry crops, are preferred. For example, wheat, rice, corn, potatoes, barley, tobacco, soybean canola, maize, oilseed flats, lilies, crested plants, orchids, irises, onions, palms, tomatoes, legumes, or Arabidopsis It can be used as a host plant. Host plants also include those that can be easily infected by infectious viruses such as nicotiana, preferably Nicotiana bentamiana, or Nicotiana cleverandii.

One feature of the invention is the use of plant viral nucleic acids containing one or more non-native nucleic acid sequences that can be transcribed in a host plant. Such a nucleic acid sequence may be a native nucleic acid sequence originating in the host plant. Preferably, such a nucleic acid sequence is a non-native nucleic acid sequence that does not normally occur in the host plant. For example, a plant viral vector may comprise more than one viral sequence, including more than one taxon-derived virus. Plant viral nucleic acids can also include non-viral sequences such as foreign genes, regulatory sequences, and fragments thereof from bacteria, fungi, plants, animals or other sources. Such foreign sequences may encode commercially useful proteins, polypeptides, or fusion products thereof, such as enzymes, antibodies, hormones, drugs, vaccines, pigments, antimicrobial polypeptides, and the like. Or they may be sequences that regulate the transcription or translation of the viral nucleic acid, package the viral nucleic acid, and promote systemic infection in the host.

In some embodiments of the invention, the plant virus vector may contain one or more additional natural or non-native subgenomic promoters capable of transcribing or expressing an adjacent nucleic acid sequence in a host plant. Such unnatural subgenomic promoters are inserted into plant viral nucleic acids without destroying the biological function of the plant viral nucleic acid using methods known in the art. For example, when transfected with plant cells, a CaMV promoter can be used. The subgenomic promoter may function in a particular host plant. For example, if the host is tobacco, TMV, a tomato mosaic virus, or other virus containing a subgenomic promoter may be used. The inserted subgenomic promoter should be compatible with the TMV nucleic acid and be capable of directing the transcription or expression of the adjacent nucleic acid sequence in the tobacco. It is particularly contemplated that two or more different non-native subgenomic promoters may be used. The non-native nucleic acid sequence may be transcribed or expressed in the host plant under the control of a subgenomic promoter to produce a nucleic acid product of interest.

In some embodiments of the invention, the recombinant plant viral nucleic acid may be modified by conventional techniques to delete all or part of the native envelope protein coding sequence or to place the native envelope protein coding sequence under control of the non-native plant virus subgenomic promoter Further modifications are possible. If deleted or otherwise inactivated, the non-native coat protein coding sequence is inserted under the control of one of the non-native subgenomic promoters, or alternatively under the control of a native coat protein gene subgenomic promoter. Thus, the recombinant plant viral nucleic acid contains a coat protein coding sequence which can be a natural or non-native coat protein coding sequence under the control of one of the natural or non-native subgenomic promoters. Natural or non-natural coat protein genes can be used in recombinant plant viral nucleic acids. As in the case of natural envelope proteins, the non-native coat protein can encapsulate the recombinant plant viral nucleic acid and provide systemic spread of the recombinant plant viral nucleic acid in the host plant.

In some embodiments of the invention, the recombinant plant viral vector is constructed to express a plant viral envelope protein and a fusion protein between the foreign gene of interest or the polypeptide of interest. Such recombinant plant viruses provide a high level of expression of the nucleic acid of interest. The location at which the viral coat protein is linked to the amino acid product of the nucleic acid of interest may be referred to as a fusion joint. The proposed product of such a construction may have one or more fused joints. The fusion junction can be located at the carboxy terminus of the viral envelope protein or at the amino terminus of the coat protein portion of the construct. When the nucleic acid of interest is located internally to the 5 'and 3' residues of the nucleic acid sequence encoding the viral coat protein, there are two fusion links. That is, the nucleic acid of interest may be located 5 ', 3', upstream, downstream, or inside the coat protein. In some embodiments of such recombinant plant viruses, a "leak" initiation or termination codon may occasionally occur in a fusion link that does not result in translation termination.

In some embodiments of the invention, the nucleic acid encoding the reporter protein or the antibiotic / herbicide resistance gene (s) can be constructed as a carrier protein for the polypeptide of interest, which can facilitate detection of the polypeptide of interest. For example, a green fluorescent protein (GFP) can be co-expressed with a polypeptide of interest. In another example, a reporter gene, beta-glucuronidase (GUS), may be used. In another example, drug resistance markers, such as those whose expression results in kanamycin resistance, can be used.

Since the RNA genome is typically infectious, place the cDNA near the appropriate promoter so that the RNA is produced in the producer cell. If the capped RNA is infectious, the RNA is capped using conventional techniques. In addition, the capped RNA can be packaged in vitro to form assembled virions with TMV-added coat protein. Such assembled virions can then be used to inoculate plants or plant tissues. Alternatively, unencapsulated RNA may also be used in embodiments of the present invention. In contrast to the techniques carried out in the scientific literature and granted patents (Ahlquist et al., U.S. Patent No. 5,466,788), unencapsulated transcripts for viral expression vectors are infectious in both plant and plant cells. Capping increases the efficiency of infection, but is not a requirement to cause infection of the viral expression vector in plants. In addition, an infectious viral vector can be constructed by inserting a nucleic acid between the transcription initiation site of the promoter and the initiation site of the cDNA of the viral nucleic acid. One or more nucleic acids can be inserted. In some embodiments of the invention, the inserted nucleic acid sequence may comprise a G at the 5 ' end. Alternatively, the inserted nucleic acid sequence may be (GNN) _x or (GTN) _x , which is GNN, GTN, or their debris.

In some embodiments of the invention, more than one nucleic acid is produced for a polymorphic virus vector construct. In this case, each nucleic acid may need its own assembly origin. Each nucleic acid could be made to contain a subgenomic promoter and a non-native nucleic acid. Alternatively, the insertion into the nucleic acid of a monomolecular virus of a non-native nucleic acid may result in the production of both nucleic acids (i. E., The nucleic acid necessary for the production of a bispecific viral vector). This would be advantageous if it would be desirable to maintain the replication and transcription or expression of the nucleic acid of interest separately from the replication and translation of part of the coding sequence of the native nucleic acid.

The recombinant plant virus nucleic acid can be prepared by cloning a viral nucleic acid. If the viral nucleic acid is DNA, it can be cloned directly into a suitable vector using conventional techniques. One technique is to attach the origin of replication to viral DNA that is compatible with the cell to be transfected. In this manner, a DNA copy of the chimeric nucleic acid sequence is produced in the transfected cell. When the viral nucleic acid is RNA, a DNA copy of the viral nucleic acid is first prepared by well-known procedures. For example, viral RNA can be transcribed into DNA using reverse transcriptase to produce subgenomic DNA fragments, and double stranded DNA can be prepared using DNA polymerase. The cDNA is then cloned into a suitable vector and cloned into the cell to be transfected. In some cases, the cDNA is first attached to a promoter that is compatible with the producer cell. The recombinant plant viral nucleic acid can then be cloned into any suitable vector compatible with the producer cell. Alternatively, the recombinant plant viral nucleic acid is inserted into a vector adjacent to a promoter that is compatible with the producer cell. In some embodiments, the vector to which the cDNA is linked can be directly transfected into the infectious RNA in vitro and inoculated into the host plant. If necessary, maps of the cDNA fragments are generated and combined into the appropriate sequences to produce a full-length DNA copy of the viral RNA genome.

Donor organisms derived from a library of sequence inserts include prokaryotes, protists, fungi, plants and animals. The prokaryote system is Archaebacteriobionta (archaebacteria): Archaebacteriophyta (methane, salt and sulfolobus); Eubacteriobionta (true bacterium) Weasel: Eubacteriophyta gate; Viroids; And Viruses. The prototype system consists of Phycobionta family: Xanthophyta 275 (yellow algae) gate, Chrysophyta 400 (yellow brown algae) gate, Dinophyta (yellow brown algae) 1,000 Bacillariophyta 5,500 (diatomaceous), Cryptophyta 74, Haptophyta 250 (attached anchovy) door, Yuglenopita Euglenophyta 550 gate, Chlorophyta gate, Chlorophyceae 10,000 green river, Charophyceae 200 river, Phaeophyta 900 (Phryophyta) Brown algae) doors, and Rhodophyta 2,500 (red algae) doors; Mastigobionta 960 Aphis: Chytridiomycota 750 door, and Oomycota 475 (water mold) door; Myxobionta 320 Aphis: 21 Acrasiomycota (cellular mucus), and Myxomycota 500 (true mucus) gates. The fungi are Zygomycota 570 (polymorphic fungus) Q: Zygomycotina armum; And Eumycota 350 (wall fungus) Ascomycotina 56,000 Basidiomycotina 25,000 Ammonium, Deuteromycotina 22,000 (bacterium) Bacillus spp. Incomplete bacteria) Ammon, and Lichenes 13,500. The plant system may be selected from the group consisting of Bryophyta, Hepatophyta, Anthocerophyta, Psilophyta, Lycophyta, Sphenophyta, Pterophyta, Coniferophyta, Cycadeophyta, Ginkgophyta, Gnetophyta, and Anthophyta. The animal kingdom is composed of Porifera, Cnidaria, Ctenophora, Platyhelminthes, Nemertea, Rotifera (rotifers), Nematoda (roundworms), Mollusca (snails, clams, squid and octopus), Onychophora (velvet beetle) Annelida, Arthropoda, Phoronida, Bryozoa, Brachiopoda (shellfishes), shellfishes (shellfishes) Echinodermata (sea urchins and starfish), and chordata (vertebrates-fish, birds, reptiles, mammals). The preferred donor organism is human. Host organisms are those that can be infected by a virus containing an infectious RNA or recombinant viral nucleic acid. Host organisms include prokaryotes, protists, fungi, and animal-derived organisms. Preferred host organisms are animal-derived organisms such as fungi and insects (e.g., C. elegans) such as yeast (e.g., S. cerevisiae).

To prepare a DNA insert containing the nucleic acid sequence of the donor organism, the first step is to construct a pool of cDNA libraries, genomic DNA libraries, or mRNAs of the donor organism. Full-length cDNA or genomic DNA can be obtained in public or private repositories. CDNA and genomic libraries of, for example, cattle, chickens, dogs, flies, fish, frogs, humans, mice, pigs, rabbits, rats, and yeast; And retroviral libraries can be obtained from Clontech (Palo Alto, Calif.). Alternatively, the cDNA library can be obtained by a method known to those skilled in the art, for example, a method of separating mRNA and transcribing mRNA to cDNA using a reverse transcriptase (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2), Vols 1-3, Cold Spring Harbor Laboratory, (1989) or Current Protocols in Molecular Biology, F. Ausubel et al., Greene Publishing and Wiley-Interscience, New York (1987) ≪ / RTI > Genomic DNA from BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome), or TAC (Transformable Artificial Chromosome, Lin et al., Proc. Natl. Acad Sci USA 96: 6535-6540 Can be obtained in public or private repositories.

Alternatively, gene pools over-expressed in tumor cell lines as compared to normal cell lines can be produced or obtained in public or private repositories. Zhang et al. (Science, 276: 1268-1272 (1997)) use a series of analysis of gene expression (SAGE) method (Velculescu et al., Cell, 88: 243 (1997)), 500 transcripts were identified. The expression of DNA that is overexpressed in a tumor cell line of a host organism can cause a change in the host organism, and such a pool of DNA is another source of DNA inserts for the present invention. BAC / YAC / TAC DNA, DNA or cDNA can be mechanically sized or cut by enzymes into smaller segments. The fragment is ligated to the adapter with the tacky end and is short-cloned into the recombinant viral nucleic acid vector. Alternatively, the fragment may be bridged end-connected to a recombinant viral nucleic acid vector. A recombinant viral nucleic acid comprising a nucleic acid sequence derived from a cDNA library or a genomic DNA library is then constructed using conventional techniques. The prepared recombinant viral nucleic acid vector contains a nucleic acid insert derived from the donor organism. The nucleic acid sequence of the recombinant viral nucleic acid is transferred from the host organism to the RNA; RNA can regulate the expression of phenotypic characteristics by positive or antisense mechanisms. Nucleic acid sequences can also modulate the expression of more than one phenotypic trait. Nucleic acid sequences from prokaryotes, protists, fungi, plants and animals can be used to assemble DNA libraries. This method can then be used to discover useful dominant gene phenotypes from DNA libraries through gene expression in host organisms.

When using plants as donor organisms, donor plants and host plants can be genetically distant or irrelevant: they can belong to different genera, families, trees, rivers, acorns, or gates. Donor plants include commercially interested plants such as edible crops, seed crops, oil crops, ornamental crops, and forestry crops. For example, wheat, rice, corn, potato, barley, tobacco, soybean canola, maize, oilseed rape, arabic acid, nicotiana can be selected as donor plants.

To prepare a DNA insert containing the nucleic acid sequence of a donor plant, the first step is typically to construct a cDNA, genomic DNA library, or RNA pool of plant of interest. The full-length cDNA can be obtained in a public or private repository, for example, a cDNA library of Arabidopsis thaliana can be obtained from the Arabidosis Biological Resource Center. Alternatively, the cDNA library can be obtained by a method known to those skilled in the art, for example, a method of separating mRNA and transcribing mRNA to cDNA using a reverse transcriptase (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2), Vols 1-3, Cold Spring Harbor Laboratory, (1989) or Current Protocols in Molecular Biology, F. Ausubel et al., Greene Publishing and Wiley-Interscience, New York (1987) Can be prepared from a wild sample. Genomic DNA from BAC (bacterial artificial chromosome), YAC (yeast artificial chromosome), or TAC (Transformable Artificial Chromosome, Lin et al., Proc. Natl. Acad Sci USA 96: 6535-6540 For example, in a public or private repository, for example, the Arabicopsis Biological Resource Center. BAC / YAC / TAC DNA or cDNA can be mechanically size-fractionated with smaller fragments or can be cleaved by enzymes. The fragment is ligated to the adapter with the tacky end and shotgun cloned into the recombinant viral nucleic acid vector. Alternatively, the fragment may be bridged end-connected to a recombinant viral nucleic acid vector. A recombinant plant viral nucleic acid comprising a nucleic acid sequence derived from a cDNA library or a genomic DNA library is then constructed using conventional techniques. The recombinant viral nucleic acid vector produced contains a nucleic acid insert derived from a donor plant. The nucleic acid sequence of the recombinant viral nucleic acid is transferred from the host plant to the RNA; RNA can regulate the expression of phenotypic characteristics by positive or antisense mechanisms. The nucleic acid sequence can also encode an expression of more than one phenotypic trait. Sequences derived from wheat, rice, corn, potato, barley, tobacco, soybean, canola, maize, oilseed rape, arabicids, and other crop species can be used to assemble DNA libraries. This method can then be used to find useful dominant gene phenotypes from DNA libraries through gene expression.

Those skilled in the art will appreciate that this embodiment is only representative of many constructions suitable for the present invention. All such constructs are contemplated and are intended to be within the scope of the invention. The present invention is not intended to be limited to any particular viral construct, but specifically contemplates the use of all operable constructs. Those skilled in the art will be able to construct plant viral nucleic acids based on molecular biology techniques well known in the art. Suitable techniques are described in Sambrook et al. (Second Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor (1989); Methods in Enzymol. (Vols. 68, 100, 101, 118, and 152-155) (1979, 1983, 1986 and 1987); And DNA Cloning, D.M. Clover, H., IRL Press, Oxford (1985); Walkey, Applied Plant Virology, Chapman & Hall (1991); Matthews, Plant Virology, 3rd edition, Academic Press, San Diego (1991); Turpen et al., J. of Virological Methods, 42: 227-240 (1993); U.S. Patent Nos. 4,885,248, 5,173,410, 5,316,931, 5,466,788, 5,491,076, 5,500,360, 5,589,367, 5,602,242, 5,627,060, 5,811,653, 5,866,785, 5,889,190 and 5,589,367, U.S. Patent Application No. 08 / 324,003. Nucleic acid manipulation and enzymatic treatment are carried out according to the manufacturer ' s recommended procedure during manufacture of such constructs.

II. Express a member of the sequence insert derived by the donor organism in the host plant

Host plants include plants of commercial interest, such as edible crops, seed crops, oil crops, ornamental crops and forestry crops. For example, wheat, rice, corn, potato, barley, tobacco, soybean canola, maize, oilseed rape, arabic acid, nicotiana can be used as a host plant. In particular, host plants that are susceptible to infection by viruses containing recombinant viral nucleic acids are preferred. Preferred host plants include Nicotiana, preferably Nicotiana bentamiana, or Nicotiana Clever Landy.

Individual clones can be transfected into host plants: 1) protoplasts; 2) plant whole; Or 3) plant tissues such as leaves of plants (Dijkstra et al., Practical Plant Virology: Protocols and Exercises, Springer Verlag (1998); Plant Virology Protocol: From Virus Isolation to Transgenic Resistance in Methods in Molecular Biology, Taylor et al., Humana Press (1998)). In some embodiments of the invention, the transfer of the plant virus expression vector into a plant is accomplished by inoculation of in vitro transcribed RNA, inoculation of virions, or inoculation of plant cells from a nuclear cDNA, It can be affected by systemic infection. In all cases, coinfection can cause rapid and rampant systemic expression of the desired nucleic acid sequence within a plant cell.

The host may be infected with recombinant viral nucleic acid or recombinant plant virus by conventional techniques. Suitable techniques include, but are not limited to, blade abrasion, polishing in solution, high-speed water spray, and other wounds of the host besides infecting the host seed with water containing recombinant viral RNA or recombinant plant virus. More specifically, suitable techniques include the following:

(a) Manual inoculation. Manual inoculation is carried out using a neutral pH, low molar concentration of phosphate buffer, with celite or carabundan (usually about 1%) added. One to four drops of the preparation are applied to the top surface of the leaves and gently rubbed.

(b) Mechanical inoculation of plant layers. Plant layer inoculation is accomplished by spraying (gas propulsion) the vector solution into the tractor driven lawnmower while cutting the leaves. Alternatively, the grass of the plant layer is cut and the vector solution is sprayed immediately on the cut leaves.

(c) Single-leaf high-pressure spray. Single plant inoculations can also be performed by spraying the leaves with a narrow, directional spray (50 psi, 6-12 inches from the leaves) containing approximately 1% carabundan in the buffered vector solution.

(d) Vacuum infiltration. The inoculation can be carried out by placing the host organism in a substantially vacuum pressure environment to facilitate infection.

(e) high-speed robot inoculation. It is particularly applicable when the organism is a plant, and individual organisms can grow in a large array such as a microtiter plate. Mechanisms such as robots can then be used to deliver the nucleic acid of interest.

(f) ballistics (high pressure gun) inoculation. Single plant inoculations can also be performed by particle impact. Impact particle delivery systems (BioRad Laboratories, Hercules, (A)) can be used to transfect plants such as Nicotiana benthamiana as previously described (Nagar et al., Plant Cell, 7: 705-719 1995).

Alternative methods of introducing viral nucleic acids into host plants include Agrobacterium infection or Agrobacterium-mediated transformation (also known as Agrobacterium infection), as described in Grimsley et al., Nature 325: 177 (1987) . This technique utilizes the common feature of Agrobacterium that fixes the plant by transferring a portion of their DNA (T-DNA) into the host cell, which is inserted into the nuclear DNA in the host cell. T-DNA is defined by a 25-bp long border sequence, and all DNA between these border sequences is also transferred into plant cells. Insertion of the recombinant plant viral nucleic acid between the T-DNA border sequences results in the transfer of the recombinant plant viral nucleic acid into the plant cell, wherein the recombinant plant viral nucleic acid is replicated within the plant cell and then spread throughout the plant. Agro-infection has been reported to be associated with potato worm virus (PSTV) (Gardner et al., Plant Mol. Biol. 6: 221 (1986); CaV (Grimsley et al., Proc. Natl. Acad Sci. USA 83: (Grimsley et al., Nature 325: 177 (1987)) and Lazarowitz, S., Nucl. Acids Res. 16: 229 (1988)); (Hayes et al., J. Gen. Virol. 69: 891 (1988)) and tomato gold mosaic virus (TGMV) (Elmer et al. Plant Mol. Biol. 10: 225 (1988) and Gardiner et al., EMBO J. 7: 899 (1988)). Agro-infections of susceptible plants could therefore be carried out as virions containing recombinant plant viral nucleic acid based on any nucleic acid sequence in the virus. Particle impact or electroporation, or any other method known in the art may also be used.

In some embodiments of the invention, the infection can also be performed by placing the nucleic acid sequence selected in an organism such as E. coli or yeast (whether inserted into the genome of the organism or not) and applying the organism to the surface of the host organism have. Such a mechanism may produce a secondary transfer of a nucleic acid sequence selected thereby into a host organism. This is a particularly practical embodiment when the host organism is a plant. Likewise, infection can be achieved by first packaging the selected nucleic acid sequence into pseudovirus. Such a method is described in WO 94/10329. While the teachings of this reference are specific to bacteria, one of ordinary skill in the art will readily recognize that the same procedure is readily adaptable to other organisms.

Plants can be grown from seed in a mixture of ^{"Peat-Lite Mix TM (Speedling} , Inc. Sun City, Fl) and Nutricote ^TM" sustained-release fertilizer 14-14-14 (Chiss-Asahi Fertilizer Co., Tokyo, Japan) . Plants can grow in a controlled environment provided 16 hours of cancer and 8 hours of cancer. Sylvania "Gro-Lux / Aquarium" Broad Spectrum 40 Watts Fluorescent Growth Lights [Osram Sylvania Products, Inc. Danvers, MA]. The temperature can be maintained at about 80 동안 during the light cycle and at about 70 동안 during the dark cycle. The humidity can be between 60 and 85%.

III. Detect phenotype or biochemical change as a result of expression

After infecting the host plant with individual clones of the library, one or more phenotypic or biochemical changes may be detected.

The phenotypic changes in the host plants can be determined by any known method in the art. Phenotypic changes may include growth rate, color, or morphological changes. Typically, such methods include visual, macroscopic, or microscopic analysis. For example, growth changes such as growth inhibition, color change (for example, leaf sulphide, stain, bleaching, yellowing) can be easily observed. Examples of morphological changes include, among others, developmental defects, wilting, and necrosis.

Biochemical changes can be determined by any analytical method known in the art for detecting, quantifying, or separating DNA, RNA, proteins, antibodies, carbohydrates, lipids, and small molecules. Selected methods may include Northern, Western blotting, MALDI-TOF, LC / MS, GC / MS, two-dimensional IEF / SDS-PAGE, ELISA, and the like. In particular, a suitable method can be carried out in a fully automated manner with a high throughput, using a robot. Examples of biochemical changes may include accumulation of substrates or products from enzymatic reactions, changes in biochemical pathways, inhibition or enhancement of endogenous gene expression in the cytoplasm of cells, changes in RNA or protein profile. For example, a clone in a viral vector library can be functionally classified based on the metabolic pathway or the resulting visible / selective phenotype that is affected in the organism. This method enables rapid determination of gene function for unknown nucleic acid sequences of donor organisms in addition to host organisms. Moreover, this method can be used to quickly identify the function of the full-length DNA of the unknown function. Functional identification of the unknown nucleic acid sequence in a library of one organism can then enable rapid identification of similar unknown sequences in expression libraries for other organisms based on sequence homology. Such information is useful in many aspects, including human medicine.

Biochemical or phenotypic changes in an infected host plant may be associated with the biochemistry or phenotype of the uninfected host plant. Alternatively, a biochemical or phenotypic change in an infected host plant is further associated with a host plant infected with a viral vector containing an unknown sequence of regulatory nucleic acid. Regulatory nucleic acids may have similar sizes, but differ in sequence from sequences of nucleic acid inserts derived from libraries. For example, if a nucleic acid insert derived from a library is found to encode a GTP binding protein in the antisense orientation, a nucleic acid derived from the gene encoding the green fluorescent protein can be used as the regulatory nucleic acid. It is known that green fluorescent proteins do not have the same effect as GTP binding proteins when expressed in host plants.

In some embodiments, the phenotype or biochemical properties can be determined by complementary analysis, i.e., by observing the inherent gene (s) whose function is replaced or expanded by introducing the nucleic acid of interest. Discussion of such phenomena is provided by [Napoli et al., The Plant Cell 2: 279-289 (1990)]. The phenotypic or biochemical characteristics include (1) analyzing biochemical changes in the accumulation of substrate or product from the enzymatic reaction according to any means known to those skilled in the art; (2) observing any changes in the biochemical pathway that can be altered in the host organism as a result of nucleic acid expression; (3) utilizing techniques known by those skilled in the art to observe the inhibition of endogenous gene expression in the cytoplasm of cells as a result of nucleic acid expression; (4) utilizing techniques known by those skilled in the art to observe changes in RNA or protein profiling as a result of nucleic acid expression; Or (5) selecting an organism that can grow or remain viable in the presence of, for example, harmful or toxic substances such as pharmaceutical ingredients.

One useful means for determining the function of a nucleic acid transfected into a host plant is to observe the effect of gene silencing. Traditionally, functional gene knockouts have been performed by the identification of conditional, homologous mutants obtained in reverse crossing followed by inactivation of transcription factors into chromosomes or random insertion of T-DNA. In this regard, some of the teachings are provided by WO 97/42210 incorporated herein by reference. As an alternative to traditional knockout analysis, the EST / DNA library of donor organisms can be assembled into a viral transcription plasmid. The nucleic acid sequence in the transcription plasmid library can then be introduced into the host cell as part of a functional RNA virus that transcribes and then silences the homologous target gene. The EST / DNA sequence may be introduced into the viral vector in either positive or antisense orientation, and the orientation may be directional or random based on the cloning strategy. Robot-based, high throughput automated cloning schemes can be used to assemble and identify libraries. Alternatively, the EST / cDNA sequence may be inserted into the genomic RNA of the viral vector so that it appears as genomic RNA during viral replication in the host cell. The library of EST clones is then inoculated into host organisms that are transcribed with infectious RNA and susceptible to viral infection. The viral RNA containing the EST / cDNA sequence conferred from the original library is now present at a sufficiently high level in the cytoplasm of the host organism cell to cause gene silencing after transcription of the endogenous gene in the host organism. Since the replication machinery of the virus produces both sense and antisense RNA sequences, the orientation of the EST / cDNA insert is generally irrelevant in that it produces the desired phenotype in the host organism.

The present invention provides a method for transiently expressing virus-positive or antisense RNA in a transfected plant. Such a method is much faster than the time required to obtain genetically engineered antisense transgenic organisms. Systemic infection and expression of viral antisense RNAs occur as short as a few days after inoculation, while it takes several months or longer to produce a single transgenic organism. The present invention provides a method for identifying a gene involved in growth regulation by inhibiting expression of a specific endogenous gene using a viral vector. The present invention provides a method for identifying a specific gene and a biochemical pathway in a donor organism or a host plant using an RNA viral vector.

It is known that silencing of an endogenous gene can be achieved as a homologous sequence from the same plant family. For example, Kumagai et al. (Proc. Natl. Acad. Sci. USA 92: 1679 (1995)) disclose that nicotianabentamina gene for phytoene unsaturation enzyme (PDS) And that it is silenced by transfection of viral RNA derived from clones containing partial tomatoes (Lycopersicon esculentum) cDNA. Kumagai et al. Demonstrate that a gene encoding a plant-derived PDS can be silenced by transfecting a host plant with a nucleic acid of known sequence from a donor plant of the same group, i.e. the PDS gene. The present invention provides a method for silencing genes in a host organism by transfecting non-plant host organisms with viral nucleic acids containing nucleic acid inserts derived from cDNA libraries or genomic DNA libraries or RNA pools of non-plant organisms. Unlike Kumagai et al., The sequence of the nucleic acid insert of the present invention need not be confirmed prior to transfection. Another aspect of the present invention is to provide a method for silencing conserved genes of non-plant systems; The antisense transcript of a biological organism results in a reduced expression of the endogenous genes of prokaryotes, protists, fungi and animal-derived host organisms. The present invention is exemplified by GTP binding proteins. In eukaryotic cells, GTP binding proteins function in a variety of cellular processes, including signal transduction, cytoskeletal tissue, and protein transport. The GTP-binding protein of low molecular weight (20-25 kilodalton) contains the ras and its close species (e.g. Ran), rho and its close species, the rab family, and the ADP-ribosomal factor (ARF) family . Heterogeneous trimer and monomeric GTP-binding proteins that may be involved in secretion and intracellular transport are subdivided into two structural classes: rab and the ARF family. Many ARFs have been isolated and identified. ARF shares structural features with both ras and trimer GTP-binding protein families. The present invention relates to a transcription of a gene encoding a GTP-binding protein, in which the gene of a plant such as Nicotiana is derived from a plant of different genus such as Arabidopsis, and a clone-derived infectious RNA containing an open reading frame of GTP- Which can be silenced by the user. The present invention also demonstrates that the GTP binding protein is highly homologous in humans, frogs, mice, cows, flies and yeast at the nucleic acid level besides the amino acid level. The present invention thus provides a method of silencing a conserved gene in a host organism by transfecting the host with an infectious RNA derived from a homologous gene of a non-plant organism.

Nucleic acid sequences that may result in a change in host phenotype include cell growth, proliferation, differentiation and development; Cell communication; And cell suicide pathways. Genes that regulate cell or organism growth include, for example, genes encoding GTP binding protein, ribosomal protein L19 protein, S18 ribosomal protein, and the like. (Or HER-2 or neu) gene is amplified and overexpressed in one third of breast, stomach, and ovarian cancers, and the ribosomal protein It is reported that mRNA encoding L19 is more abundant in breast cancer samples expressing high levels of erbB-2. Lijsebettens et al. (EMBO J., 13: 3378-3388 (1994)) report that in Arabidopsis, mutations in PFL cause the first perforated leaf, reduced biomass and growth retardation. PFL encodes the ribosomal protein S18, which is highly homologous to the rat S18 protein. Genes involved in cell or organism development include, for example, genes encoding a G-protein-associated receptor protein such as a homeobox-containing gene and a rhodopsin family. The homeobox gene is a family of regulatory genes that contain a common 183 nucleic acid sequence (homeobox) and that encode a specific nuclear protein (homeoprotein) that acts as a transcription factor. The homeobox sequence itself encodes the homeome domain, a 61 amino acid domain, involved in the recognition and binding of sequence-specific DNA motifs. The specificity of this binding allows home proteins to activate or inhibit expression of a single target gene downstream. Homeobox, initially identified in genes controlling Drosophila development, was subsequently isolated from evolutionarily distant animal species, plants, and fungi. Some indications suggest involvement of the homeobox gene in the regulation of cell growth, and in cancer development when abnormal regulation (Cillo et al., Exp. Cell Res., 248: 1-9 (1999)). Other nucleic acid sequences that may result in alterations in the organism include genes encoding receptor proteins such as hormone receptors, cAMP receptors, serotonin receptors, and the calcitonin family of receptors; And light regulatory DNA encoding Leu zipper motifs (Zheng et al., Plant Physiol. 116: 27-35 (1998)). Cell growth, proliferation, differentiation and development; Cell communication; And de-regulation or alteration of apoptotic pathway processes can lead to cancer. Therefore, identifying the nucleic acid sequences involved in the process and determining their function is beneficial to human therapy; It also provides tools for cancer research.

A library of human nucleic acid sequences is cloned into the vector. The vector is applied to the host to infect. Each infected host is grown with uninfected host and Nulve vector infected host. Nul Vectors will not show phenotypic or biochemical changes other than the effects of the virus itself. Each host is observed daily for visible differences between the infected host and its two controls. Identify traits in each host showing observable phenotypic or biochemical changes. Identify donor nucleic acid sequences, obtain full length gene sequences, and obtain full length genes in the host if the host genes are associated with traits. The nucleotide sequences of the two genes are determined and their homology is determined. A variety of biochemical tests can also be performed in the host or host tissue depending on the information desired. A variety of phenotypic changes or characteristics and biochemical tests are described herein. The functional gene profile can be obtained by repeating the process several times.

A large amount of DNA sequence information is being generated as a share, which can start a database with which it is related. It is possible to link between sequences from various species that are expected to perform similar biochemical or regulatory functions. It is also possible to link between the expected enzymatic activity and the biochemical and regulatory pathways present. Likewise, anticipated enzymes or regulatory activities and known small molecule inhibitors, activators, substrates or substrate analogs can be linked. Phenotypic data from expression libraries expressed in transfected hosts can be automatically linked within such related databases. You can quickly find genes with similar predictive roles of interest in other organisms.

The present invention also relates to a method for altering the phenotype or biochemistry of a plant by transiently expressing a nucleic acid sequence of a donor plant in a host plant antisense direction, which inhibits gene expression inherent in the mitotic tissue of the host plant. One or more phenotypic or biochemical changes in the host plant are detected by the methods described previously. Transient expression of a nucleic acid sequence in a host plant can affect gene expression in the mitotic tissue. Split tissue is of interest in plant development, since plant growth is driven by the formation and activation of mitotic tissue throughout the entire life cycle. The present invention is exemplified by the nucleic acid sequence encoding the ribosomal protein S18. The activity of the S18 promoter is restricted to the mitotic tissue (Lijsebettesn et al., EMBO J. 13: 3378-3388). Transient expression of the nucleic acid sequence in the host plant can cause signaling to the mitotic tissue and can affect gene expression in the mitotic tissue.

One problem with gene silencing in host plants is that many plant genes are multigene families. Therefore, effective silencing of gene function can be particularly problematic. However, according to the present invention, the nucleic acid can be inserted into the viral genome and effectively silence the function of a specific gene function or a multigene family. It is currently believed that about 20% of the plant genes are multigene families.

A detailed discussion of some aspects of the " gene silencing " effect is provided in co-pending patent application, U.S. Patent Serial No. 08 / 260,546 (WO 95/34668, published on Dec. 22, 1995), the disclosure of which is incorporated herein by reference do. Through interaction of the inhibitory RNA with the target mRNA that occurs in the cytoplasm and / or nucleus of the cell, the RNA can reduce the expression of the target gene.

EST / cDNA libraries of plants such as Arabidopsis thaliana can be assembled into the plant virus transcription plasmid background. The cDNA sequence in the transcription plasmid library can then be introduced into plant cells as cytoplasmic RNA to transcribe and silence the endogenous gene. The EST / cDNA sequence may be introduced into the plant viral transcription plasmid in one direction (or both directions) of the positive or antisense orientation, and the orientation may be either directional or random, based on the cloning strategy. The library can be assembled using a high throughput automated cloning strategy using robots. An EST clone can be inserted after the subgenomic promoter replicated to appear as a subgenomic transcript during viral replication in plant cells. Alternatively, the EST / cDNA sequence can be inserted into the genomic RNA of the plant virus vector so that it appears as genomic RNA during viral replication in plant cells. The library of EST clones is then inoculated into host plants transcribed with infectious RNA and susceptible to viral infection. The viral RNA containing the EST / cDNA sequence conferred from the original library is now present in a high enough concentration in the cytoplasm of the host plant cell to cause post-transcriptional gene silencing of the gene in the host plant. Since the replication machinery of the virus produces both sense and antisense RNA sequences, the orientation of the EST / cDNA insert is normally independent of producing the desired phenotype in the host plant.

The present invention also relates to a method for producing a gene encoding a GTP binding protein derived from rice, barley, maize, soybean, maize, oilseed, and other plants of commercial interest, using another gene having homology with the gene to be isolated A method for isolating a conserved gene such as a gene. Libraries containing full length cDNAs of donor plants such as rice, barley, corn, soybeans and other important crops can be obtained from shared and personal sources or can be prepared from plant mRNA. The cDNA is inserted into a viral vector or small subcloning vector such as pBluescript (Strategene), pUC18, M13, or pBR322. The transformed bacteria are then blotted and individual clones are selected by standard methods. Bacterial transformants or DNA are rearranged at high density on membrane filters or glass slides. The full-length cDNA encoding the GTP-binding protein can be identified by probing the filter or slide with a labeled probe made from DNA encoding a GTP-binding protein from a labeled nucleic acid insert or Arabidopsis resulting in a change in the host plant. Useful labels include radioactive, fluorescent, or chemiluminescent molecules, enzymes, and the like.

Alternatively, genomic libraries containing sequences from rice, barley, corn, soy, and other important crops can be obtained from shared and personal sources, or can be prepared from plant genomic DNA. BAC clones containing the entire plant genome were constructed and organized in minimal redundant order. Individual BACs are sheared into sections and cloned directly into viral vectors. Clones that fully encompass the entire BAC form the BAC viral vector sub-library. Genomic clones can be identified by probing a filter containing BAC with a labeled probe made from DNA encoding a GTP binding protein from a labeled nucleic acid insert or Arabidopsis resulting in a change in the host plant. Useful labels include radioactive, fluorescent, or chemiluminescent molecules, enzymes, and the like. BACs that hybridize to the probe are selected and their corresponding BAC viral vectors are used to generate the infectious RNA. Plants transfected with the BAC sub-library are screened for changes in function, e. G. Growth rate or changes in color. When a change in function is observed, the insert of such a clone or corresponding plasmid DNA is identified by dideoxy sequencing. This provides a rapid method of obtaining a genomic sequence for a plant protein, e. G., A GTP binding protein. Using this method, once a DNA sequence has been identified in one plant, such as Arabidopsis thaliana, it can be used to identify conserved sequences of similar functions present in other plant libraries.

Functional genomic screens are initiated using the tobacco mosaic virus TMV-O coat protein capsid for the infection of Nicotiana benthamiana, a plant associated with a common tobacco plant. The cDNA library for Arabidopsis thaliana was obtained from the Arabidopsis Biology Resource Center, The phage mid vector is recovered by Not I cleavage. The cDNA is transformed into a plasmid. The plasmid is transcribed into viral vector RNA. The insert is antisense orientation as shown in the figure until all the cDNAs in each cDNA library appear in the viral vector. Each viral vector is sprayed with enough force on the leaves of the two-week old Nicotiana benthamiana or host plant to cause tissue injury and local viral infection. Each infected plant is grown along with the infected plant as a non-infected plant and control as a null insert vector. All plants are grown in an artificial environment with a 16 hour light cycle and an 8 hour cancer cycle. The lumens are about the same in each plant. Visible and photographic observations of the phenotype are performed at intervals of two days, and each infected plant and each of its control groups are recorded and compared. The data is stored in the Laboratory Information Management System database. Plants that are inhibited from growing at the end of the observation period are classified for analysis. The nucleic acid insert contained in the viral vector clone 740AT # 120 is associated with severe growth arrest of one of the plants. The nucleotide sequence of clone 740AT # 120 is determined. The base sequence of the phage animal from the host plant is also determined. Clone 740AT # 120 appeared to have 80% homology with the host plant nucleic acid sequence. The homology of the amino acid sequence is 96%. The entire cDNA sequence of the insert was obtained by sequencing and was found to encode a GTP binding protein. The host plant animal is selected and the base sequence is determined. It also encodes a GTP-binding protein. We conclude that this GTP-binding protein coding sequence is highly conserved in nature. This information is useful for drug development besides toxicological studies.

A complete classification system of gene functionality for fully sequenced eukaryotes was established for yeast. This classification system can be modified for plants and divided into appropriate categories. Such an organizational structure can be used to quickly identify herbicide target sites that can confer a lethal phenotype of dominance, thereby being useful in helping to design reasonable herbicide programs.

The present invention also relates to a method for increasing the yield of cereal crops. [Rice Biotechnology Quarterly 37: 4 (1999) and Ashikari et al., Proc. Natl. Acad. Sci. USA 96: 10284-10289 (1999), transgenic rice plants transformed with the rgpl gene coding for rice GTP-binding protein were shorter than control plants but produced more seeds than control plants Reported. In order to increase the yield of grain crops, the method involves transiently expressing the nucleic acid sequence of the donor plant in a grain crop antisense direction, which results in growth inhibition and increased seed production of the grain crop do. A preferred method comprises the steps of cloning a nucleic acid sequence into a plant viral vector and infecting the crop with a recombinant viral nucleic acid containing said nucleic acid sequence. Preferred plant virus vectors are derived from brom mosaic virus, rice necrosis virus, or gemini virus. Preferred grain crops include rice, wheat, and barley. The nucleic acid expressed in the host plant contains, for example, the GTP binding protein open reading frame, which is antisense orientation. The method provides transient expression of the gene to obtain a plant inhibited from growth. As less energy is invested in plant growth, more energy is available for the production of seeds, which increases crop yields. The method has advantages over other methods using transgenic plants because it does not affect the genome of the host plant.

In order to provide a more clear and consistent understanding of the specification and claims, including the categories given in such terms herein, the following definitions are provided:

Adjacent: the position of the nucleic acid sequence that is 5 'or 3' close to the defined sequence. Generally, contiguous means within 2 or 3 nucleotides of the relevant site.

Antisense Inhibition: A type of gene regulation based on cytoplasmic, nuclear or organelle inhibition of gene expression due to the presence of RNA molecules in cells that are complementary to at least a portion of the mRNA to be translated. It is specifically contemplated that the RNA molecule may be one of an RNA virus or an mRNA from a host cell genome or DNA virus.

Cell culture: a proliferating group of cells that can be one of undifferentiated or differentiated, growing adjacent or non-adjacent.

Chimeric sequence or gene: A nucleic acid sequence derived from two or more heterologous portions. The sequence may contain DNA or RNA.

Encoding sequence: A ribonucleic acid sequence that results in the formation of a cell polypeptide when transcribed and translated or merely translated, resulting in the formation of a cell polypeptide, or a ribonucleic acid or ribonucleic acid sequence when translated.

Compatibility: Ability to work with other components of the system. A vector or plant or animal viral nucleic acid that is compatible with the host can be replicated in the host. A coat protein that is compatible with the viral nucleic acid sequence is capable of encapsulating the viral sequence.

Complementarity analysis: As used herein, the term refers to observing changes in the organism when introducing the nucleic acid sequence into the organism, after deletion or mutagenesis of the selected gene, so that it no longer functions well in its normal role . A gene that is complementary to a deleted or mutated gene can restore the genetic phenotype of the selected gene.

Dual heterologous subgenomic promoter expression system (DHSPES): A positive strand RNA vector with a dual heterologous subgenomic promoter expression system for increasing, decreasing, or altering the expression of a protein, peptide or RNA, preferably its initiation For example, those described in U.S. Patent Nos. 5,316,931, 5,811,653, 5,589,367, and 5,866,785, which are incorporated herein by reference.

Expressed sequence tag (EST): A relatively short single-pass DNA sequence obtained from cDNA clones and at least one end of RNA derived therefrom. They may be in either the 5 'or 3' direction. EST appears to be useful in identifying specific genes.

Expression: As used herein, the term is meant to include the incorporation of one or more transcription, reverse translation and translation.

Functional gene profile: A set of genes of an organism that encode biochemical or phenotypic characteristics. The functional gene profile of an organism is found by screening the nucleic acid sequence from the donor organism by overexpression or inhibition of the gene in the host organism. The functional gene profile requires a collection or library of nucleic acid sequences from the donor organism. The functional gene profile will depend on the ability of the library or collection of donor nucleic acids to cause overexpression or inhibition in the host organism. Thus, the functional gene profile will depend on the amount of donor gene that can be over expressed or suppressed in the host gene or that can be expressed in the host organism in the absence of the homologous host gene.

Gene: A separate nucleic acid sequence that serves to produce one or more cell products and / or perform one or more intercellular or intracellular functions.

Gene silencing: reduction of gene expression. The viral vector expressing the gene sequence of the host may induce gene silencing of the homologous gene sequence.

Homology: The degree of nucleic acid similarity in all or part of a gene sequence sufficient to cause gene suppression when the nucleic acid sequence is delivered in antisense orientation.

Host: A cell, tissue or organism capable of replicating a nucleic acid, such as a vector or viral nucleic acid, and capable of being infected by a virus, including a viral vector or viral nucleic acid. The term is intended to include, where appropriate, prokaryotic and eukaryotic cells, organs, tissues or organisms. Bacteria, fungi, yeast, and animals (cells, tissues, or organisms) are examples of hosts.

Infection: The ability of a virus to deliver nucleic acid to a host or to introduce a viral nucleic acid into a host, where the viral nucleic acid is replicated, the virus protein is synthesized, and new virus particles are assembled. In the present context, the terms " propagation " and " infectious " are used interchangeably herein. The term also includes the ability of the selected nucleic acid sequence to be inserted into the genome, chromosome or gene of the target organism.

Insert: A range of nucleic acid sequences, typically over 20 bp in length.

Multigene family: A set of genes transmitted by replication and mutation from some ancestral genes. Such genes may be clustered together on the same chromosome or may be dispersed on different chromosomes. Examples of multigene families include those encoding histones, hemoglobin, immunoglobulins, histocompatibility antigens, actin, tubulin, keratin, collagen, heat shock proteins, saliva glue proteins, chorionic proteins, corpus proteins, egg yolk proteins, do.

Non-native: Any RNA or DNA sequence that does not normally appear in cells or organisms where it is located. Examples include recombinant viral nucleic acids and genes or ESTs contained therein. That is, the RNA or DNA sequence may be non-canolaic for viral nucleic acid. Such RNA or DNA sequences do not naturally appear in viral nucleic acids. In addition, the RNA or DNA sequence may be non-ternary to the host organism. That is, such RNA or DNA sequences do not appear naturally in the host organism.

Nucleic acid: As used herein, the term is intended to include any DNA or RNA sequence of full gene sequence sub-size at one or more nucleotides. The term is intended to include all nucleic acids that occur naturally in a particular cell or organism or that appear non-naturally in a particular cell or organism.

The nucleic acid of interest: the term is intended to refer to the nucleic acid sequence whose function is to be determined. Sequences are normally non-linear with respect to viral vectors, but may be natural or non-natural to the host organism.

Phenotypic characteristics: observable, measurable or detectable properties due to the expression or inhibition of the gene (s).

Plant cells: Structural and physiological units of plants, consisting of protoplasts and cell walls.

Plant organ: a distinct and visible part of a plant, such as a root, stem, leaf or stomach.

Plant tissue: Any tissue of a plant in a plant or culture. The term is intended to encompass any group of whole plants, plant cells, plant organs, protoplasts, cell cultures, or plant cells organized in structural and functional units.

Positive sense inhibition: a type of gene regulation based on cytoplasmic inhibition of gene expression due to the presence of RNA molecules that are substantially homologous to at least a portion of the mRNA to be translated.

Promoter: A 5'-flanking, unencrypted sequence substantially contiguous to the coding sequence associated with the initiation of transcription of the coding sequence.

Protoplast: isolated plant or bacterial cell without some or all of the cell wall.

Recombinant viral nucleic acid: A viral nucleic acid modified to include a non-native nucleic acid sequence. Such unnatural nucleic acid sequences may be any biologically or purely synthetic, but they may also include nucleic acid sequences that occur naturally in the organism into which the recombinant viral nucleic acid is introduced.

Recombinant virus: a virus containing a recombinant viral nucleic acid.

Subgenomic promoter: Promoter of subgenomic mRNA of viral nucleic acid.

Substantial sequence homology: Represents nucleic acid sequences that are substantially functionally equivalent to each other. Nucleic acid differences between such sequences having substantial sequence homology are not important when they affect the function of the gene product or RNA encoded by such sequence.

Systemic infection: refers to infection through a substantial portion of an organism, including a diffusion mechanism, including transport from one infected cell to a nearby or distant additional cell in addition to mere direct cell inoculation.

Transient expression: expression of a nucleic acid sequence in a host without the insertion of a nucleic acid sequence into the host genome, such as via a viral vector.

Transposon: A nucleic acid sequence, such as a DNA or RNA sequence, that can be moved or moved within a gene, chromosome, or genome.

The present invention provides a method for detecting the presence of a property in a host plant by expressing a nucleic acid sequence derived from a donor organism in a antisense or positive sense direction in a host plant. Once the presence of the property is confirmed by phenotypic changes, the nucleotide sequence of the nucleic acid insert in the cDNA clone or vector is determined. The present invention provides a rapid method of determining the presence of a property in a host plant by screening for phenotypic or biochemical changes in a transfected host plant as a nucleic acid sequence of a donor organism and a method for identifying the nucleic acid sequence and its function.

Summary of the Invention

The present invention relates in principle to: (1) introducing into a viral vector a library of sequence inserts derived by a host organism in a positive or antisense orientation; (2) expressing each insert in a host plant, and (3) detecting phenotypic or biochemical changes in the host plant as a result of expression. Host plants can be terminal or dicot plants, plant tissues or plant cells. Donor organisms include species derived from prokaryotes, protists, fungi, plants or animals, such as humans, mice, fruit flies, and the like. If the donor organism is also a plant, the donor plant and the host plant typically belong to different genus, tree, tree, river, moon, or gate. The function of a sequence insert in a library is typically unknown. The number of sequence inserts in the library is typically greater than about 10, 15, 20, 50, 100, 200, 500, 1000, 5000, or 15,000, The length of each insert is typically longer than about 50, 100, 200, or 500 base pairs.

More particularly, the invention relates to a method of altering the phenotype or biochemistry of a host plant, a method of determining phenotypic or biochemical changes in a host plant, and a method of determining the presence of a property in a host plant. The method comprises transiently expressing a nucleic acid sequence of a donor organism in a host plant in the antisense or positive sense direction, identifying the change in the host plant, and correlating the phenotype or biochemical change with sequence expression. The nucleic acid sequence does not need to be isolated, identified, or characterized prior to transfection with the host organism.

The present invention also relates to a method of transiently expressing a nucleic acid sequence library in a host organism, determining a phenotypic or biochemical change in the host plant, identifying characteristics associated with the change, identifying the donor gene associated with the trait, Identifying a homologous host gene and annotating the sequence as a phenotype or function associated therewith.

The present invention also relates to a method for determining the function of a nucleic acid sequence, including a gene in a donor organism, by transfecting the nucleic acid sequence in a host plant in a manner to effect phenotypic or biochemical changes in the host plant. In one embodiment, the recombinant viral nucleic acid is prepared to include a donor nucleic acid insert. The recombinant viral nucleic acid produces antisense or positive sense RNA in the cytoplasm that infects the host plant and causes a reduced or increased expression of the cellular gene that is inherent in the host organism. Once the presence of the property is confirmed by phenotypic or biochemical changes, the function of the nucleic acid is determined. Nucleic acid inserts in cDNA clones or vectors are then sequenced. The nucleic acid sequence is determined by standard sequence analysis.

One aspect of the present invention is a method for identifying and determining a nucleic acid sequence in a donor organism wherein the function of the sequence is to introduce the nucleic acid into the host plant via a viral nucleic acid suitable for expressing the nucleic acid in the transfected plant, The function of the inherent gene is lost. This method utilizes the principle of post-transcriptional gene silencing of an inherent host genetic animal, such as antisense RNA, or positive sense RNA. In particular, this silencing function is useful for silencing multigene families in donor organisms. In addition, overexpression of positive sense RNA, which results in overproduction of the protein, can lead to phenotypic or biochemical changes in the host.

Another aspect of the present invention is to find a gene in a donor organism having the same function as a gene in a host plant. The method begins by constructing a pool of cDNA libraries, or genomic DNA libraries, or RNAs of donor organisms from, for example, tissues or cells of a human, mouse, or fruit fly. The recombinant viral nucleic acid containing the nucleic acid insert derived from the library is then prepared and used to infect the host plant. Infected host plants are examined for phenotypic or biochemical changes. Recombinant viral nucleic acids that result in phenotypic or biochemical changes in the host plant are identified and the sequence of the nucleic acid insert is determined by standard methods. Such a nucleic acid sequence in a donor organism may have substantial sequence homology to the sequence in the host plant, for example the nucleic acid sequence is conserved between the donor and the host plant. Once the nucleic acid sequence is determined, it can be labeled and used as a probe to isolate the full-length cDNA from the donor organism. The present invention provides a rapid means of identifying the function and sequence of a donor bio-nucleic acid; Information that expands so rapidly can then be used in the field of genomics.

Another aspect of the invention relates to a method for increasing the yield of grain crops. The method involves transiently expressing a nucleic acid sequence of a donor plant in the antisense or positive sense direction in a grain crop, which results in inhibited growth and increased seed production of the grain crop. A preferred method involves the step of cloning a nucleic acid sequence into a plant viral vector and infecting the crop with a recombinant viral nucleic acid containing said nucleic acid sequence.

Another aspect of the invention is to find genes with the same function in different plants. The method starts with the first plant cDNA, genomic DNA library, or RNA pool. The recombinant viral nucleic acid containing the nucleic acid insert derived from the library is then prepared and used to infect different host plants. Infected host plants are examined for phenotypic or biochemical changes. Identify recombinant viral nucleic acids that cause phenotypic or biochemical changes in host plants and determine the sequence of nucleic acid inserts by standard methods. Such a nucleic acid sequence in the first plant has substantial sequence homology with the sequence of the host plant: the nucleic acid sequence is conserved between the two plants. The present invention provides a rapid means of revealing the function and sequence of a nucleic acid of interest; Such rapidly expanding information can then be used in the field of genomics.

Another aspect of the invention is the production of human proteins in host plants. After isolating and identifying nucleic acids of similar function from human and host plants, the amino acid sequences derived from the DNA are compared. The plant nucleic acid sequence is changed to encode the same amino acid sequence as the human protein. The nucleic acid sequence may be changed according to any conventional method such as site-directed mutagenesis or polymerase-based DNA synthesis.

Host plants include commercially interested plants such as edible crops, seed crops, oil crops, ornamental crops, and forestry crops. For example, wheat, rice, corn, potato, barley, tobacco, soybean canola, maize, oilseed rape, arabic acid, Nicotiana can be selected as host plants. In particular, host plants that can be infected by viruses, including recombinant viral nucleic acids, are preferred.

The plant virus vector may contain a natural or non-native subgenomic promoter, a coat protein coding sequence, and one or more non-native nucleic acid sequences. Some viral vectors used in accordance with the present invention may be encapsulated by a coat protein encoded by a recombinant virus. The recombinant viral nucleic acid can be replicated in the host plant and can cause phenotypic or biochemical changes in the host plant by transcription or expression of the non-native nucleic acid. Any suitable vector construct useful for causing local or systemic expression of a nucleic acid in a host plant is within the scope of the present invention.

The following examples further illustrate the present invention. This embodiment is for illustrative purposes only and should not be construed as limiting the same.

Example 1

Construction of Arabidopsis thaliana cDNA library in double subgenomic promoter vector

The Arabidopsis thaliana cDNA library was obtained from the Arabidopsis Biology Resource Center (ABRC). Four libraries from ABRC were size-sorted by inserts of 0.5-1 kb (CD4-13), 1-2 kb (CD4-14), 2-3 kb (CD4-15), and 3-6 kb (CD4-16). All libraries are high quality and used by dozens of families to isolate genes. PBluescript from Lambda ZAP II vector The phageomide was cut in bulk and the library was recovered as a plasmid according to standard procedures.

Alternatively, cDNA inserts in CD4-13 (Lambda ZAP II vector) were digested with NotI and recovered. In most cases, cleavage into NotI favored a complete Arabidopsis thaliana cDNA insert because the original library was assembled as a NotI adapter. NotI is an 8-base cutter that rarely cleaves plant DNA. To insert the NotI fragment into the transcription plasmid, the pBS735 transcription plasmid (Figure 1) was digested with PacI / XhoI and generated from the oligonucleotides 5'-TCGAGCGGCCGCAT-3 '(SEQ ID NO: 1) and 5'-GCGGCCGC-3'Lt; / RTI > adapter DNA sequence. The resulting plasmid, pBS740 (Figure 2) contains one NotI restriction site for bi-directional insertion of the NotI fragment from the CD4-13 library. The recovered colonies were collected on a Qiagen BioRobot 9600 From this for preparation of plasmid minis. The BioRobot 9600 Lt; RTI ID = 0.0 > 96-well < / RTI > format and obtain DNA of transcriptional quality. The Arabidopsis cDNA library was transformed into plasmids and analyzed by agarose gel electrophoresis to identify clones with inserts. Clones with inserts were transplanted in vitro and inoculated into Nicotiana benthamiana or Arabidopsis thaliana. Leaf disks selected from transfected plants were then taken for biochemical analysis.

Example 2

Genomic DNA library construction in recombinant viral nucleic acid vector

Genomic DNA appearing in the BAC (bacterial artificial chromosome) or YAC (yeast artificial chromosome) library was obtained from the Arabidopsis Biology Resource Center (ABRC). The BAC / YAC DNA was mechanically size-fractionated, ligated to an adapter with an adherend end, and cloned into a recombinant viral nucleic acid vector. Alternatively, the mechanically sized genomic DNA is blunt end ligated into the recombinant viral nucleic acid vector. The recovered colonies were collected on a Qiagen BioRobot 9600 To prepare plasmid mini. BioRobot 9600 Lt; RTI ID = 0.0 > 96-well < / RTI > format and obtained DNA of transcriptional quality. The recombinant viral nucleic acid / Arabidopsis genomic DNA library is analyzed by agarose gel electrophoresis (template quality control step) to identify clones with inserts. Clones with inserts were transplanted in vitro and inoculated into Nicotiana benthamiana and / or Arabidopsis thaliana. Leaf disks selected from transfected plants were then taken for biochemical analysis.

The genomic DNA of Arabidopsis typically contains a 2.5 kb (kilo base) gene on average. Genomic DNA fragments of 0.5 to 2.5 kb obtained by random shearing of DNA were shorthornly assembled into a recombinant viral nucleic acid expression / knockout vector library. Given that the genome size of Arabidopsis is approximately 120,000 kb, the randomized recombinant viral nucleic acid genomic DNA library needs to include 48,000 independent inserts of at least 2.5 kb in size to achieve a single range of Arabidopsis genome. Alternatively, a randomized recombinant viral nucleic acid genomic DNA library needs to include 240,000 independent inserts of at least 0.5 kb in size to achieve a single range of Arabidopsis genome. Assembling the recombinant viral nucleic acid expression / knockout vector library from the genomic DNA rather than the cDNA has the potential to overcome the known difficulties encountered when attempting to clone a rare and low frequency mRNA in a cDNA library. Recombinant viral nucleic acid expression / knockout vector libraries prepared with genomic DNA will be particularly useful as gene silencing knockout libraries. In addition, the dual heterologous subgenomic promoter expression system (DHSPES) expression / knockout vector library prepared with genomic DNA would be particularly useful for the expression of intronless genes. Moreover, other plant species with medium to small genomes (e. G., Roses, approximately 80,000 kb) would be particularly useful for recombinant viral nucleic acid expression / knockout vector libraries prepared with genomic DNA. Recombinant viral nucleic acid expression / knockout vector libraries can be prepared from existing BAC / YAC genomic DNA or newly prepared genomic DNA for any plant species.

Example 3

Construction of a genomic DNA or cDNA library in the DHSPES vector, and transfection of the individual clones of the vector library to T-DNA markers or transposons labeled or mutated plants

Construction of a genomic DNA or cDNA library in a recombinant viral nucleic acid vector and transfection of the individual clones of said vector library into T-DNA labeled or transposon labeled or mutated plants are carried out as described in the procedures described in Examples 1 and 2 . Such procedures can be readily devised to complement mutations introduced by random insertion mutagenesis of T-DNA sequences or transposon sequences.

Example 4

Construction of Nicotiana bentamiana cDNA library

Nutrient Nicotiana botendamiana plants were harvested at 3.3 weeks after sowing and sliced into three groups of tissues: leaves, stems and roots. Each tissue group was instantaneously frozen in liquid nitrogen and total RNA was isolated from each group separately using the following hot borate method. The frozen tissue was ground into fine powder with pre-cooled mortar and pestle, and then homogenized in a further pre-cooled glassy tissue mill. Immediately thereafter, 2.5 ml / g tissue was added to hot (~ 82 ° C) XT buffer (0.2 M borate decahydrate, 30 mM EGTA, 1% (w / v) SDS). The pH was adjusted to 9.0 with 5 N NaOH, treated with 0.1% DEPC and autoclaved. Before use, 1% deoxycholate (sodium salt), 10 mM dithiothreitol, 15 Nonidet P-40 (NP-40) and 2% (w / v) polyvinylpyrrolidone, MW 40,000 (PVP- Was added to the milled tissue. Tissues were homogenized for 1-2 minutes and quickly poured into pre-cooled Oak Ridge centrifuge tubes containing 105 μl of 20 mg / ml proteinase K in DEPC-treated water. The tissue mill was rinsed with an additional 1 ml of hot XT buffer per g tissue, and this buffer was then added to the remaining homogenate. The homogenates were incubated at 100 rpm, 42 [deg.] C for 1.5 hours. 2M KCl was added to the homogenate to a final concentration of 160mM and the mixture was incubated on ice for 1 hour to precipitate the protein. The homogenate was centrifuged at 12,000 x g for 20 minutes at 4 DEG C, and the supernatant was filtered through sterile Miraclose through a clean 50 mL Oak Ridge centrifuge tube. 8M LiCl was added to a final concentration of 2M LiCl and incubated on ice overnight. The precipitated RNA was collected by centrifugation at 12,000 x g for 20 minutes at 4 ° C. The pellet was washed three times in 3-5 ml of 4 ° C 2M LiCl. Each time the pellet was resuspended with a glass rod and then centrifuged at 12,000 x g for 20 minutes at 4 ° C. The RNA pellet was suspended in 2 ml 10 mM Tris-HCl (pH 7.5) and purified from insoluble cell components by centrifugation at 12,000 x g for 20 min at 4 [deg.] C. RNA containing the supernatant was transferred to a 15 ml Corex tube and precipitated overnight at -20 ° C with 2.5 volumes of 100% ethanol. RNA was pelleted by centrifugation at 9,800 x g for 30 minutes at 4 < 0 > C. The RNA pellet was washed in 1-2 ml of cold 70% ethanol and centrifuged at 9,800 x g for 5 minutes at 4 < 0 > C. Residual ethanol was removed from the RNA pellet under vacuum, and the RNA was resuspended in 200 μl DEPC-treated secondary distilled water and transferred to a 1.5 ml centrifuge tube. Corex tubes were rinsed in 100 占 퐇 DEPC treated secondary distilled water and then added to the remaining RNA. The RNA was then precipitated with 1/10 volume of 3M sodium acetate, pH 6.0 and 2.5 volumes of cold 100% ethanol at -20 ° C for 1-2 hours. The tubes were centrifuged at 16,000 x g for 20 minutes, and the RNA pellet was washed with cold 70% ethanol and centrifuged at 16,000 x g for 5 minutes. After drying the pellet under vacuum, the RNA was resuspended in DEPC-treated water. This is total RNA.

Messenger RNA was purified from total RNA using the Poly (A) Pure kit (Ambion, Austin TX) following the manufacturer's instructions. The reverse transcription reaction was used to synthesize cDNA from the mRNA template using a Stratagene (La Jolla, CA) or Gibco BRL (Gaithersburg, MD) cDNA cloning kit. For the Stratagene library, the cDNA was subcloned into the bacteriophage at the EcoRI / XhoI site by linking arms using the Gigapack III Gold kit (Stratagene, La Jolla, Calif.) According to the manufacturer's instructions. For the Gibco BRL library, cDNA was subcloned into tovomovirus vectors containing the fusion of TMV-U1 and TMV-U5 at the NotI / XhoI site.

Example 5

Expression of Chinese cucumber cDNA clone pQ21D in plants transfected in the positive sense direction confirms that it encodes alpha-tricosanthin.

We have developed a plant virus vector that expresses alpha-tricoxanthin in transfected plants. From the genomic clone SEO, the open reading frame (ORF) for alpha-trichosanthin was located under the control of the TMV envelope protein subgenome promoter. TTU51A Infectious RNA from QSEO # 3 (FIG. 3: nucleic acid sequence as SEQ ID NO: 2 and amino acid sequence as SEQ ID NO: 3) was prepared by in vitro transcription using SP6 DNA-dependent RNA polymerase, I use it to mechanically inoculate my child. Hybrid viruses spread to all non-inoculated top leaves, as confirmed by local damage infectivity assays and PCR amplification. Viral symptoms consist of mild yellowing and systemic leaf distortion and plant growth inhibition. 27 kDa alpha-tricoxanthin accumulated on the upper leaf (14 days after inoculation) and cross-reacted with anti-tricosanthin antibody.

Plasmid construction

The 0.88 kb XhoI, AvrII fragment containing the alpha-trichosanthin coding sequence was amplified from the genomic DNA isolated from Trichosanthes kirilowii Maximowicz by the oligonucleotide QMIX: 5'-GCC TCG AGT GCA GCA TGA TCA GAT (SEQ ID NO: 4) and Q1266A 5'-TCC CTA GGC TAA ATA GCA TAA CTT CCA CAT CAA AGC-3 '(downstream) (SEQ ID NO: 5) And amplified by induction. The alpha-tricoxanthin open reading frame was identified by dideoxy sequencing and was located under the control of the TMV-U1 envelope protein subgenomic promoter by subcloning into TTU51A to generate the plasmid TTU51A QSEO # 3.

Analysis of in vitro transcription, inoculation, and transfected plants

Nicotiana benthamiana plants were inoculated with the in vitro transfectants of KpnI-cleaved TTU51A QSEO # 3. Virion was isolated from infected Nicotiana benthamiana leaf with TTU51A QSEO # 3 transcript.

Purification, < RTI ID = 0.0 > immunological < / RTI > detection, and in vitro testing of alpha-

At 2 weeks after inoculation, the total soluble protein was separated from the upper non-inoculated Nicotiana benthamiana leaf tissue and assayed for cross reactivity against alpha-tricoxanthin antibody. Systemically infected tissue proteins were analyzed on 0.1% SDS / 12.5% polyacrylamide gels and transferred to nitrocellulose membranes by electroblotting for 1 hour. The blotted membranes were treated with goat anti-alpha-trichosanthin antiserum for 2000 hr dilution for 1 hour. Increased chemiluminescence mustard radish peroxidase linked rabbit anti-goat IgG assays (Cappel Laboratories) were performed according to the manufacturer's instructions (Amersham). The blotted film was exposed for 10 seconds. The shorter luminescent exposure times and the longer chemiluminescent exposure times of the blotted films gave the same quantitative results.

Example 6

Expression of the bell pepper cDNA in the plant transfected in the positive sense direction confirms that it encodes capsanthin-capsorubin synthase.

The biosynthesis of leaf carotenoids in Nicotiana bentamiana was altered by reordering the synthetic pathways of non-natural color-specific xanthophyll capsanthin using RNA viral vectors. The cDNA encoding capsanthin-capsorubin synthase (Ccs) was placed under the transcriptional control of the tovomovirus subgenomic promoter. The leaves of transfected plants expressing Ccs exhibited orange phenotype and accumulated high levels of capsanthin. This phenomenon is associated with a reduction in tilacoid membrane distortion and granny stacking. In contrast to the predominant situation in color variants, capsaicin was not esterified and its increased level was balanced by simultaneous reduction of major leaf xanthophylls, suggesting self-regulation of chloroplast carotenoid composition. Capsanthin is exclusively supplemented with light-harvesting complexes of pomegranate & monomer II. The proof that higher plant antenna complexes can accommodate non-natural carotenoids provides compelling evidence for functional remodeling of photosynthetic membranes by proper design of carotenoids.

Construction of Ccs expression vector . One XhoI, AvrII site was amplified by polymerase chain reaction (PCR) using the oligonucleotides: 5'-GCCTCGAGTGCAGCATGGAAACCCTTCTAAAGCTGTAATCCCTTCTAAAGCTTTTCC-3 '(upstream) (SEQ ID NO: 6), 5'-TCCCTAGGTCAAAGGCTCTCTATTGCTAGATTGCCC-3' (Ccs) cDNA by pepsin mutagenesis in the cytoplasmic capsaicin-capsorubin synthase (Ccs) cDNA. The 1.6 kb XhoI, AvrII cDNA fragment was placed under the control of the TMV-U1 envelope protein subgenomic promoter by subcloning into TTO1A, and the plasmid TTO1A CCS + (Figure 4: nucleic acid sequence as SEQ ID NO: 8 and Amino acid sequence as SEQ ID NO: 9).

Carotenoid analysis. On the 12th day after inoculation, the upper leaves were harvested from 12 plants and lyophilized. The resulting non-saponified extract was evaporated to dryness under argon and weighed to determine the total lipid content. Pigment analysis from total lipid content was performed by HPLC and also by thin layer chromatography on silica gel G using hexane / acetone (60:40 (V / V)). Plants transfected with TTO1A CCS + accumulated high levels of capsanthin (36% of total carotenoids).

Example 7

Expression of cDNA Encoding Tomato Phytoene Synthetic Enzyme and Phytoene Unsaturated Enzyme in the Positive and Antisense Direction in Nicotiana bentamiana

Isolation of Tomato Mosaic Virus cDNA. The 861 bp fragment (5524-6384) from the putative coat protein subgenomic promoter, coat protein gene, and tomato mosaic virus (fruit necropsy line F; tom-F) containing the 3 'end was amplified with primers 5'-CTCGCAAAGTTTCGAACCAAATCCTC-3' Upstream) (SEQ ID NO: 10) and 5'-CGGGTACCTGGGCCCCAACCGGGGGTTCCGGGGG-3 '(downstream) (SEQ ID NO: 11) and subcloned into the HincII site of pBluescript KS-. A hybrid virus consisting of TMV-U1 and ToMV-F was constructed by exchanging the 874 bp BamHI-KpnI ToMV fragment with pBGC152 to generate the plasmid TTO1. The inserted fragment was confirmed by dideoxynucleotide sequencing. One AvrII site was amplified by PCR mutagenesis using the following oligonucleotides: 5'-TCCTCGAGCCTAGGCTCGCAAAGTTTCGAACCAAATCCTCA-3 '(upstream) (SEQ ID NO: 12), 5'- CGGGGTACCTGGGCCCCAACCGGGGGTTCCGGGGGTTCCGGGGGGTTCCGGGGGG-3' (downstream) At the downstream of the XhoI site of the plasmid TTO1A.

Separation of cDNA encoding tomato phytoene synthase and partial cDNA encoding tomato phytoene unsaturation. The following oligonucleotide: PSY, 5'-TATGTATGGTGCAGAAGAACAGAT-3 '(upstream) (SEQ ID NO: 14), 5'-AGTCGACTCTTCCTCTTCTGGCATC-3' (downstream) (SEQ ID NO: 15) from tomato fruit RNA aging partial cDNA PDS, 5'-TGCTCGAGTGTGTTCTTCAGTTTTCTGTCA-3 '(upstream) (SEQ ID NO: 16), 5'-AACTCGAGCGCTTTGATTTCTCCGAAGCTT-3' (downstream) (SEQ ID NO: 17). Approximately 3 x 10 ⁴ colonies from the Lycopersicon esculentum cDNA library were screened by colony hybridization using the ³² P-labeled tomato phytoene synthase PCR product. Hybridization was performed at 42 ° C for 48 hours in 50% formamide, 5X SSC, 0.02M phosphate buffer, 5X Denhart's solution, and 0.1mg / ml sheared female calf thymus DNA. Filters were washed at 65 ° C in 0.1X SSC, 0.1% SDS before radiometry. PCR products and phytoene synthase cDNA clones were identified by nucleotide sequencing of dideoxynucleotides.

DNA sequencing and computer analysis. The PstI and BamHI fragments containing the phytoene synthetase cDNA and the partial phytoene-unsaturated enzyme cDNA were ligated into pBluescript KS + (Stratagene, La Jolla, Calif.). Nucleic acid sequence determination of KS + / PDS # 38 and KS + / 5'3'PSY was performed by dideoxy termination using a single stranded template (Maniatis, Molecular Cloning, First Edition). Nucleotide sequence analysis and amino acid sequence comparison were performed with PCGENE And DNA Inspector IIE program.

Construction of Tomato Phytoene Synthetic Enzyme Expression Vector. The XhoI fragment containing the cDNA of the tomato phytoene synthase was subcloned into TTO1. Vector TTO1 / PSY + (Figure 5: nucleic acid sequence as SEQ ID NO: 18 and amino acid sequence as SEQ ID NO: 19) contained phytoene synthase cDNA in the positive direction under the control of the TMV-U1 envelope protein subgenomic promoter; The vector TTO1 / PSY- contains phytoene synthase cDNA in antisense orientation.

Construction of viral vectors containing partial tomato phytoenetra unsaturated cDNA. The XhoI fragment containing the partial tomato phytoene-unsaturated enzyme cDNA was subcloned into TTO1. The vector TTO1A / PDS + (Figure 6) contains the phytoene-unsaturated enzyme cDNA in the positive orientation under the control of the TMV-U1 envelope protein subgenomic promoter; The vector TTO1A / PDS- contains the phytoene-unsaturated enzyme cDNA in the antisense orientation.

TTO1 / PSY +, TTO1 / PSY-, TTO1 / PDS +, TTO1 / PDS-. Infectious RNA from TTO1 / PSY +, TTO1 / PSY-, TTO1 / PDS +, and TTO1 / PDS- was prepared by in vitro transcription using SP6 DNA-dependent RNA polymerase as previously described (Dawson et al., Proc. Natl. Acad. Sci. USA 85: 1832 (1986)) and used to mechanically inoculate nicotianabentamia. Hybrid viruses spread to all inoculated upper leaves as confirmed by transmission electron microscopy, local damage infectivity assays, and polymerase chain reaction (PCR) amplification. Viral symptoms due to infection consisted of plant growth inhibition with systemic leaf twisting and mild yellowing. The leaves of the plants transfected with TTO1 / PSY + changed to orange and accumulate high levels of phytoene, while those transfected with TTO1 / PDS + and TTO1 / PDS- turned white. Northern blot analysis of agarose gel electrophoresis and virion RNA of PCR cDNAs isolated from virion RNA indicates that the vector is maintained in an extrachromosomal state and no detectable intramolecular rearrangement has occurred.

Purification and analysis of carotenoids from transfected plants. Carotenoids were separated from infected tissues systemically and analyzed by HPLC chromatography. The carotenoids were extracted from ethanol and eluted with 25-cm Spherisorb (R) solution using acetonitrile / methanol / 2-propanol (85: 10: 5) ODS-15-m column by their peak residence time and absorption spectrum. They had the same retention time as the synthetic phytoene standard and the beta-carotene standard of carrot and tomato. Nicotiana bentamiana phytonene peak transfected with TTO1 / PSY + had maximum optical absorbance at 276, 285, and 298 nm. Plants transfected with phytoene synthase encoded by the virus showed a 10-fold increase in phytoene compared to levels of uninfected plants. Expression of sense and antisense RNAs to partial phytoene-unsaturated enzymes in transfected plants increased levels of phytoene and altered biochemical pathways; It inhibited the synthesis of colored carotenoids and changed systemically infected leaves to white. HPLC analysis of these plants revealed that they also accumulated phytoene. White leaf phenotypes were also observed in plants treated with the herbicide Norflurazone, which specifically inhibits phytoene unsaturation.

At the level of phytoene this change represents one of the largest increases in any carotenoid (secondary metabolite) in any genetically engineered plant. Plants transfected with phytoene synthase encoded by the virus with a positive sense showed a 10-fold increase in phytoene compared to levels in the uninfected plants. In addition, the accumulation of phytoene in plants transfected with antisense phytoene-unsaturated enzymes suggests that viral vectors can be used as powerful means of manipulating pathways in the production of secondary metabolites through cytoplasmic antisense inhibition. The leaves of systemically infected TTO1A / PDS + plants also accumulated phytoene and showed a white pigment phenotype; The actual mechanism of inhibition is unclear. These data are described in Kumagai et al., Proc. Natl. Acad. Sci. USA 92: 1679-1683 (1995).

Example 8

Expression of phytoene-unsaturated enzymes in transfected plants using multimeric viral vectors

Construction of terminal viral vectors. BSMV is a trimer RNA virus that infects many agriculturally important terminal lobes such as oats, wheat and barley (McKinney and Greeley, " Biological characteristics of barley stripe mosaic virus strains and their evolution ", Technical Bulletin US Department of Agriculture 1324 1965). An expression vector derived from barley stripe mosaic virus (BSMV) was constructed by modifying BSMV gamma cDNA (Gustafson et al., Virology 158 (2): 394-406 (1987)) (Fig. 7A). In this example, we developed a terminal viral vector that leads to the expression of nucleic acid sequences in transfected plants. The foreign insert may be located under the control of the gamma b subgenomic promoter. Infectious BSMV gamma cDNA (gamma 42) was modified by site-directed mutagenesis. Nucleotide 5098-5103 of gamma 42 was replaced by NheI site. Using the polymerase chain reaction (PCR) mutagenesis, a 646 bp NheI fragment containing the zeomycin resistance gene was cloned into the oligonucleotides 5'-TATGCTAGCTGATTAATTAAGTCGACGAGCTGATTTAACAAATTTTAAC-3 '(upstream) (SEQ ID NO: 20) and 5'-TATGCTAGCTGAGCGGCCGCGCACGTGTCAGTCCTGCTCCTCGG-3 Was amplified from pZErO (Invitrogen Corporation, Carlsbad, Calif., USA) using (downstream) (SEQ ID NO: 21) and inserted into the Nhe site of BSMV gamma cDNA. This produces two plasmids, gamma bbst.P / N-zeo (positive orientation) and gamma bst.N / P-zeo (negative direction), in which the PacI and NotI sites are in contact with the zeomycin resistance gene (Fig. 7B).

(Petty et al., Virology 179 (2): 712-8 (1990)) were used to amplify most of the envelope protein ORF by PCR mutagenesis to improve the expression of gamma subgenomic RNA1 . The 423 bp fragment was amplified from? 42 SpI using oligonucleotides 5'-GGAAAGCCGGCGAACGTGGCG-3 '(upstream) (SEQ ID NO: 22) and 5'-TATATTCGAATCTAGAATCGATGCTAGCTTGCATGCTGTGAAGTGGTAAAAGAAATGC-3' (downstream) NgoMIV and BstBI sites. This construct only contains the untranslated portion of the envelope protein ORF required for the expression of the < RTI ID = 0.0 > oRNA < / RTI > ORF (Figure 7C).

Construction of Terminal Viral Vectors Containing Partial Maize Phytoene Enzymes. The partial cDNA encoding the phytoene unsaturation enzyme (PDS) was amplified from the corn leaf tissue RNA by the oligonucleotide pair 175 5'-ATATTAATTAACATGGACACTGGCTGCCTGTC-3 '(upstream) (SEQ ID NO: 24) encoding the PDS Met ¹ -Leu ²⁹⁰ and 180 5 5 '-ATATTAATTAACAAGGTAGCTGCTTGGAAGGATG-3' (upstream) (SEQ ID NO: 26) and 178 5'-TATGCGGCCGCCTAGCAGGTTACTGACATGTCTGC-3 '(downstream) (SEQ ID NO: 25) encoding PDS Lys ¹³⁴ -Cys ⁴³¹ (downstream) (SEQ ID NO: 27), and PDS Gln ²⁸⁴ -Ser pair to encrypt the ^{571 179 5'-ATATTAATTAACCAGTGCATTTTGATTGCTTTG-3 '} ( upstream) (SEQ ID NO: 28) and 176 5'-TATGCGGCCGCCTAAGATGGGACGGGAACTTCTCC-3' (downstream) (SEQ ID NO: No. 29) was used for amplification by RT-PCR. The 0.8 Kb amplified Pac I and Not I fragments containing the partial cDNA encoding maize PDS were amplified with Pac I and Not I fragments of < RTI ID = 0.0 > ybst.P / N-zeo and y.yb.st.N / Lt; / RTI > sub-genomic promoter by subcloning into the < RTI ID = 0.0 > I < / RTI > This removes the myelin resistance gene and cleaves it in the positive direction ([gamma] [beta] st.P / N-mPDS-N, [gamma] mPDS-C) and the negative direction ([gamma] [beta] st.P / N-mPDS-N, [gamma] ) To generate plasmids with PDS inserts.

Analysis of barley plants transfected with y.y.b.st.P / N-mPDS. Infectious BSMV RNA from cDNA clones was prepared by in vitro transcription using T7 DNA-dependent RNA polymerase (Ambion) as previously described (Petty et al., Virology 171 (2): 342-9 (1989)). Transcripts of each of the three BSMV genomes were mixed at a 1: 1: 1 ratio. A 7 [mu] l aliquot of the transcription mixture was combined with 40 [mu] l of FES and applied directly to the 12-day old bovine plant. BSMV :: mPDS hybrid viruses spread on uninoculated leaves. Early virus symptoms (1-7 days post-vaccination) due to PDS containing the constructs showed symptoms similar to wild-type BSMV infection. At 8-10 days after inoculation, BSMV-PDS plants began to show patches of striated and strangely white tissue. The affected areas were found in plants treated with NorfluRAZONE, a chemical inhibitor of PDS, which lacked necrosis or dryness, often associated with BSMV-induced bleaching, and more bleached tissue. Although the most extensive area of bleaching was generally found in infected plants with BSMV containing PDS in the sense direction, this white stripe was observed to some extent in all BSMV :: mPDS infected plants.

Purification and analysis of carotenoids from transfected barley plants. Carotenoids were isolated from 50 mg of systemically infected leaf tissue on day 18 after inoculation and analyzed by HPLC chromatography. The carotenoids were extracted in the dark in methanol and their peak residence time on a Zorbax 4.6 X 15 cm C-18 column using acetonitrile / methanol / 2-propanol (85: 10: 5) as eluent at a flow rate of 2 ml / And absorption spectrum. They had the same retention time as the synthetic phytoene standard and the beta-carotene standard of tomato and carrot. Expression of sense and antisense RNAs in partial corn phytoene - unsaturated enzymes in transfected barley inhibited the synthesis of colored carotenoids and changed systemically infected tissues to white. HPLC analysis of these plants revealed that they also accumulated phytoene. White leaf phenotypes were also observed in barley plants treated with herbicide norfluracone, which specifically inhibits phytoene unsaturation. Phytoenes extracted from barley transfected with BSMV-PDS were analyzed by HPLC, which had a residence time similar to that of the phytoene standard and showed a 10- to 60-fold increase over the level of BSMV transfected control plants Respectively.

Our findings that phytoene accumulates in transgenic barley plants with partial antisense and positive sense phytoene-unsaturated enzymes can be used to manipulate the biosynthetic pathway in the terminal lobe through cytoplasmic inhibition of the gene expression inherent in the plant virus vector .

Example 9

Expression of the bacterial CrtB gene in plants transfected in the positive sense direction confirms that it encodes phytoene synthase.

We have shown that the tobacco mosaic virus strain U1 (TMV-U1) (Goell et al., Proc. Natl. Acad. Sci. USA 79: 5818-5822 (1982)) and tobacco mild green mosaic virus (TMGMV; U5 strain) , 177: 553-8 (1990)). An open reading frame (ORF) (Armstrong et al., Proc. Natl. Acad. Sci. USA 87: 9975-9979 (1990)) against Erwinia herbicola phytogen synthetase (CrtB) Mosaic virus (TMV) coat protein subgenomic promoter. This construct also contains a gene encoding a chloroplast target peptide (CTP) for a small subunit of ribulose-1, 5-bisphosphate carboxylase (RUBISCO) (O'Neal et al., Nucl. Acids Res 15: 8661-8677 (1987)) was named TTU51 CTP CrtB as shown in Figure 8 (nucleic acid sequence as SEQ ID NO: 30 and amino acid sequence as SEQ ID NO: 31). Infectious RNA was prepared by in vitro transcription using SP6 DNA-dependent RNA polymerase (Dawson et al., Proc. Natl. Acad. Sci. USA 83: 1832-1836 (1986)); Susek et al., Cell 74: 787-799 (1993)) and used to mechanically inoculate nicotianabentamia. Hybrid viruses were spread on all uninoculated upper leaves and confirmed by local damage infectivity assays and PCR amplification. The leaves of plants transfected with TTU51 CTP CrtB exhibited systemic spreading orange pigment during plant growth and viral replication.

The leaves of the plants transfected with the TTU51 CTP CrtB had a reduction in chlorophyll content exceeding the slight reduction normally observed during viral infection (results not shown). Since previous studies suggest that the carotenoid and chlorophyll biosynthetic pathways are interconnected (Susek et al., Cell 74: 787-799 (1993)), we decided to compare the rate of synthesis of phytoene to chlorophyll. At 2 weeks after inoculation, the chloroplasts of the transfected plants with the TTU51 CTP CrtB transcript were isolated and assayed for enzyme activity. The ratio of phytoene synthase to chlorophyll synthase was 0.55 in transfected plants and 0.033 in uninoculated plants (control). The phytoene synthase activity of plants transfected with TTU51 CTP CrtB was assayed using isolated chloroplasts and labeled [ ¹⁴ C] geranyl geranyl PP. There was greatly increased phytoene and unidentified C ₄₀ alcohol in CrtB plants.

Phytoene synthase assay.

The chloroplasts were prepared as previously described (Camara, Methods Enzymol. 214: 352-365 (1993)). The phytoene synthetase assay was carried out by adding [ ¹⁴ C] geranyl geranyl PP (100,000 cpm) (prepared using pepper GGPP synthase expressed in Escherichia coli), 1 mM ATP, 5 mM MnCl ₂ , 1 mM MgCl ₂ , Triton X-100 (20 mg per chloroplast protein) and a chloroplast suspension equivalent to 2 mg protein (0.5 ml final volume). After incubation at 30 ° C for 2 hours, the reaction product was extracted with chloroform methanol (Camara, supra) and TLC on a silica gel plate developed with benzene / ethylacetate (90/10) followed by radiometric autoradiography.

Chlorophyll synthase assay

For the chlorophyll synthase assay, isolated chloroplasts were degraded by osmotic shock prior to incubation. (0.2 ml, final) consisting of [ ¹⁴ C] geranyl geranyl PP (100,000 cpm), 5 mM MgCl ₂ , 1 mM ATP, and 50 mM Tris-HCl (pH 7.6) containing a ruptured plasmid suspension equivalent to 1 mg protein Volume) was kept at 30 DEG C for 1 hour. The reaction products were analyzed as previously described.

Plasmid construction

As shown in Fig. 8, a phytoene synthase expression vector, TTU51 CTP CrtB, directed to the chloroplast was constructed in several subcloning steps. (SEQ ID NO: 32) and CrtB P300 5'-AAG ATC TCT CGA (upstream) (SEQ ID NO: 32) and nucleotide CrtB MIS 5'-CCA AGC TTC TCG AGT GCA GCA TGC AGC AAC CGC CGC TGC TTG AC- (PCR) mutagenesis using the GCT AAA CGG GAC GCT GCC AAA GAC CGG CCG G-3 '(downstream) (SEQ ID NO: 33) to the Erwinia herbicola phytoene synthase gene (Saiki et al., Science 230: 1350-1354 (1985)). The CrtB PCR fragment was ligated with pBluescript (Stratagene) to generate the plasmid pBS664. Subsequently, the 938 bp SphI, XhoI CrtB fragment from pBS664 was subcloned into a vector containing a sequence encoding the Nicotiana tabacum chloroplast target peptide (CTP) for the small subunit of RUBISCO to generate plasmid pBS670. Next, the tovomovirus vector, TTU51, was constructed. A 1020 bp fragment from tobacco soft green mosaic virus (TMGMV; U5 strain) containing the viral subgenomic promoter, coat protein gene, and 3 'end was inserted into TMGMV primer 5'-GGC TGT GAA ACT CGA AAA GGT TCC GG-3' (SEQ ID NO: 34) and 5'-CGG GGT ACC TGG GCC GCT ACC GGC GGT TAG GGG AGG-3 '(downstream) (SEQ ID NO: 35) and isolated by PCR at the HincII site of pBluescript KS- Subcloning and confirmed by dideoxynucleotide sequencing. This clone contains naturally occurring duplications of 147 bases, including the entire upstream pseudoknot domain in the 3 'unencrypted region. The hybrid virus cDNA composed of TMV-U1 and TMGMV was constructed by exchanging the 1 kb XhoI-KpnI TMGMV fragment into TTO1 (Kumagai et al., Proc. Natl. Acad. Sci. USA 92: 1679-1683 (1995)), plasmid TTU51 Respectively. Finally, a 1.1 kb XhoI CTP CrtB fragment from pBS670 was subcloned into XhoI of TTU51 to generate the plasmid TTU51 CTP CrtB. As a CTP negative control, a 942 bp Xho I fragment containing CrtB gene from pBS664 was subcloned into TTU51 to generate plasmid TTU51 CrtB # 15.

Example 10

Identification of nucleic acid sequences involved in the regulation of plant growth by cytoplasmic inhibition of gene expression in the positive sense direction using virus-induced RNA

In this embodiment, we describe a method for producing positive sense RNA using (1) an RNA viral vector, (2) a method for producing a sense RNA derived from viruses in the cytoplasm, (3) A method for increasing or inhibiting the expression of an inherent plant protein in a plant, and (4) a method for producing a transfected plant containing a virus positive sense RNA; Such a method is faster than the time required to obtain a genetically engineered sense transgenic plant. Systemic infection and expression of virus-positive sense RNAs occurs as short as four days after inoculation whereas it takes several months or more to produce a single transgenic plant. This example demonstrates that a novel positive strand viral vector, which replicates only in the cytoplasm, can be used to identify genes involved in the regulation of plant growth by increasing or inhibiting the expression of specific endogenous genes. This example also allows the identification of specific gene and biochemical pathways in plants transfected using RNA viral vectors.

Tovamovirus vectors have been developed for heterologous expression of nucleic acid sequences that have not been identified in plants transfected. A partial Arabidopsis thaliana cDNA library was placed under the transcriptional control of the tovarmovirus subgenomic promoter in the RNA viral vector. Colonies from the transformed E. coli were automatically taken using a Flexys robot and transferred to a 96 well flat bottom block containing 50 [mu] g / ml of terrific broth (TB) Amp. Approximately 2000 plasmid DNAs were separated from the overnight culture using BioRobot and infectious RNA from 430 independent clones was directly applied to the plants. At 1-2 weeks after inoculation, the transfected Nicotiana benthamiana plants were visually monitored for changes in growth rate, morphology, and color. One set of plants transfected with 740 AT # 2441 was heavily inhibited from growing. DNA sequencing revealed that this clone contained an Arabidopsis Ran GTP binding protein open-cell unique (ORF) in the positive sense direction. This demonstrates that the episomal RNA viral vector can be used to deliberately alter the metabolic pathway and cause plant growth arrest. In addition, our results show that Arabidopsis positive sense transcripts can cause phenotypic changes in Nicotiana bentamiana.

Construction of Arabidopsis thaliana cDNA library from RNA virus vector

The Arabidopsis thaliana or CD4-13 cDNA library was digested with NotI. A DNA fragment of 500 to 1000 bp was isolated by trough extraction and subcloned into the NotI site of pBS740. Escherichia coli C600 water-soluble cells were transformed with the pBS740 AT library, and colonies containing the Arabidopsis cDNA sequence were selected on 50 μg / ml of LB Amp. Recombinant C600 cells were automatically harvested using a Flexys robot and then transferred to a 96 well flat bottom block containing 50 [mu] g / ml of terrific broth (TB) Amp. Approximately 2000 plasmid DNAs were isolated from overnight cultures using BioRobot (Qiagen) and infectious RNA from 430 independent clones was directly applied to the plants.

Isolation of Gene Encoding GTP Binding Protein

At 1-2 weeks after inoculation, the transfected Nicotiana benthamiana plants were visually monitored for changes in growth rate, morphology, and color. Plants transfected with 740 AT # 2441 (Figure 9) were heavily inhibited from growing. The plasmid 740 AT # 2441 contains the TMV-U1 open reading frame (ORF) encoding the 126-, 183- and 30-kDa proteins, the TMV-U5 coat protein gene (U5 cp), the T7 promoter, the Arabidopsis thaliana CD4-13 NotI fragment, and a portion of the pUC19 plasmid. The TMV-U1 subgenomic promoter located within the negative strand of the 30 kDa ORF regulates the synthesis of CD4-13 subgenomic RNA.

DNA sequencing and computer analysis

The 841 bp NotI fragment of 740 AT # 2441 containing the Ran GTP binding protein cDNA was identified (Figure 10: nucleic acid sequence and amino acid sequence as SEQ ID NOs 36 and 37 respectively). Nucleic acid sequence determination of 740 AT # 2441 was performed using double stranded template And was performed by dideoxy termination. Nucleic acid sequence analysis and amino acid sequence comparison were performed using the DNA Strider, PCGENE and NCBI Blast programs. 740 AT # 2441 contained an open reading frame (ORF) of positive orientation, encoding a protein of 221 amino acids with an apparent molecular weight of 25,058 Da. Protein mass was calculated using a Voyager program (Perceptive Biosystems). Figure 11 shows the nucleic acid sequence alignment of 740 AT # 2441 (SEQ ID NOS: 38 and 39, respectively) for AF017991, an Arabidopsis thaliana salt stress inducible small GTP-binding protein Ran1. Figure 12 shows nucleic acid alignments of 740 AT # 2441 (SEQ ID NOS: 40 and 41, respectively) for Lichtmannata tachumum ras-like GTP binding protein L16787. Figure 13 shows amino acid comparison (SEQ ID NOS: 42 and 43, respectively) of 740 AT # 2441 to tobacco Ran-B1 GTP binding protein.

The Arabidopsis thaliana cDNA was cloned into Arabidopsis thaliana cDNA library using high-phasic images of Arabidopsis thaliana, tomato (L. esculentum), tobacco (Nicotiana tabacum), L. japonicus and V. faba GTP binding protein cDNA (99 to 82%) (Table 1). The nucleic acid sequence of 740 AT # 2441 encodes a protein with strong homology (100 to 95%) to Arabidopsis thaliana, tomato, tobacco, and soy GTP binding protein (Table 2).

The # 2441 DNA also shows a high degree of homology (67-83%) to human, yeast, mouse, and Drosophila GTP binding protein cDNAs (Table 3). The protein also has 67% -97% identity to yeast, mammals such as humans, and 79% -98% positivity (Table 4).

MALDI-TOF analysis of Nicotiana bentamiana transfected with 740 AT # 2441

Ten days after inoculation, the phyllotaxis, leaves and stems of Nicotiana benthamiana transfected with 740 AT # 2441 were frozen in liquid nitrogen. Soluble proteins were extracted in crushing buffer (100 mM Tris, pH 7.5, 2 mM EDTA, 1 mM PMSF, 10 mM BME) using a mortar and pestle. The homogenate was filtered through four layers of cheese cloth and centrifuged at 10,000 x g for 15 minutes. The supernatant was then centrifuged at 100,000 x g for 1 hour. A 500 μl aliquot of the supernatant was mixed with 500 μl 20% TCA (acetone / 0.07% BME) and stored at 4 ° C overnight. The supernatant was analyzed by MALDI-TOF (Karas et al., Anal. Chem. 60: 2299-2301 (1988)). The results showed that the soluble protein contained a newly expressed protein with a molecular weight of 25,058.

Isolation of Arabidopsis thaliana GTP-binding protein genomic clones

Genomic clones encoding the Arabidopsis thaliana GTP binding protein can be isolated by probing a filter containing arabicitabine thaliana or BAC clones using a ³² P-labeled 740 AT # 2441 NotI insert. Other members of the Arabidopsis thaliana ARF multigene family were identified using the program of the Genetics Computer Group of the University of Wisconsin.

Example 11

Identification of nucleic acid sequences involved in the regulation of plant growth by cytoplasmic inhibition of gene expression in antisense orientation using virus-induced RNA (GTP binding protein)

In this example, we describe a method for producing antisense RNA using (1) an RNA viral vector, (2) a method for producing antisense RNA induced by a virus in the cytoplasm, (3) A method for inhibiting the expression of an inherent plant protein, and (4) a method for producing a transfected plant containing a virus antisense RNA, which method is useful for obtaining a genetically engineered antisense transgenic plant It is faster than time. Systemic infection and expression of viral antisense RNA occurs several days after inoculation, while it takes several months to produce a single transgenic plant. This example demonstrates that a novel positive strand viral vector that replicates in the cytoplasm can be used to identify genes involved in the regulation of plant growth by inhibiting the expression of specific endogenous genes. This example identifies specific gene and biochemical pathways in plants transfected using RNA viral vectors.

Tovamovirus vectors have been developed for heterologous expression of nucleic acid sequences that have not been identified in plants transfected. A partial Arabidopsis thaliana cDNA library was placed under the transcriptional control of the tovarmovirus subgenomic promoter in the RNA viral vector. Colonies from the transformed E. coli were automatically taken using a Flexys robot and transferred to a 96 well flat bottom block containing 50 [mu] g / ml of terrific broth (TB) Amp. Approximately 2000 plasmid DNAs were separated from the overnight culture using BioRobot and infectious RNA from 430 independent clones was directly applied to the plants. At 1-2 weeks after inoculation, the transfected Nicotiana benthamiana plants were visually monitored for changes in growth rate, morphology, and color. One set of plants transfected with 740 AT # 120 was severely inhibited from growing. DNA sequencing revealed that this clone contained an Arabidopsis GTP binding protein open-cell unique (ORF) in the antisense orientation. This demonstrates that the episomal RNA viral vector can be used to deliberately alter the metabolic pathway and cause plant growth arrest. In addition, our results suggest that the Arabidopsis antisense transcript may stop the expression of nicotianabentamia gene.

Construction of Arabidopsis thaliana cDNA library in RNA virus vector

The Arabidopsis thaliana or CD4-13 cDNA library was digested with NotI. DNA fragments of 500 to 1000 bp were isolated by trough extraction and subcloned into the NotI site of pBS740. Escherichia coli C600 water-soluble cells were transformed with the pBS740 AT library and colonies containing Arabidopsis cDNA sequences were selected on 50 μg / ml of LB Amp. Recombinant C600 cells were automatically harvested using a Flexys robot and then transferred to a 96 well flat bottom block containing 50 [mu] g / ml of terrific broth (TB) Amp. Approximately 2000 plasmid DNAs were isolated from overnight cultures using BioRobot (Qiagen) and infectious RNA was directly applied to plants from 430 independent clones.

Isolation of Gene Encoding GTP Binding Protein

At 1-2 weeks after inoculation, the transfected Nicotiana benthamiana plants were visually monitored for changes in growth rate, morphology, and color. Plants transfected with 740 AT # 120 (Figure 14) were heavily inhibited from growing. Plasmid 740 AT # 120 includes TMV-U1 126,183, and 30 kDa ORF, TMV-U5 coat protein gene (U5 cp), T7 promoter, Arabidopsis thaliana CD4-13 NotI fragment, and part of pUC19 plasmid. The TMV-U1 subgenomic promoter located within the negative strand of the 30 kDa ORF regulates the synthesis of CD4-13 antisense subgenomic RNA.

DNA sequencing and computer analysis

A 782 bp NotI fragment of 740 AT # 120 containing ADP-ribosylation factor (ARF) cDNA was identified. The DNA sequence (774 bp) of the NotI fragment of 740 AT # 120 is as follows: 5'-

Nucleic acid sequence determination of 740 AT # 120 was performed by dideoxy termination using a double stranded template. Nucleic acid sequence analysis and amino acid sequence comparison were performed using the DNA Strider, PCGENE and NCBI Blast programs. 740 AT # 120 contained an open reading frame (ORF) in the antisense orientation, encoding a protein of 181 amino acids with an apparent molecular weight of 20,579 Da.

Sequence comparison

Figure 15 shows a nucleic acid sequence comparison of Arabidopsis thaliana 740 AT # 120 and Arabidopsis thaliana est AA042085 (SEQ ID NOs 45 and 46, respectively). The nucleic acid sequence of 740 AT # 120 was also compared to the rice (Oryza sativa) ADP ribosylating factor AF012896 (SEQ ID NOs: 47 and 48) (Figure 16); This shows 82% (456/550) positive and identifiable.

The nucleotide sequence of 740 AT # 120 represents a high degree of homology (81-84% identity and affinity) with rice, barley, carrot, maize and arabicidus tallia DNA encoding ARF, (71-84% identity and positive) with mammalian DNA such as insects such as insects, flies, amphibians such as frogs, cows, humans, and mice (Table 5).

The amino acid sequence derived from 740 AT # 120 represents a higher degree of homology (96-98% identity and 97-98% positive) with ARF from rice, carrot, maize and Arabidopsis thaliana, (61-98% identity and 78-98% positive; higher than nucleic acid sequence homology) with ARFs from mammals such as insects, cows, humans, .

The high homology of DNA encoding GTP-binding proteins from yeast, plants, insects, humans, mice, and amphibians suggests that DNA from one donor organism can be transfected into another host organism, Indicating that it can be silenced.

The 120P, a protein encoded by 740 AT # 120, has three conserved domains: the phosphate binding loop motif, GLDAAGKT (SEQ ID NO: 49), (common sequence GXXXXGKS / T, SEQ ID NO: 50); G 'motif, DVGGQ (SEQ ID NO: 51), (common sequence DXXGQ, SEQ ID NO: 52), sequence interacting with gamma-phosphate of GTP; And the G motif NKQD (SEQ ID NO: 53), (common sequence NKXD, SEQ ID NO: 54) specific for guanidinyl linkages. 120P contains an estimated glycine-myristoylation site at position 2, a potential N-glycosylation site (NXS) at position 60, and several putative serine / threonine phosphorylation sites.

Humanized DNA

The nucleic acid sequence of 740 AT # 120 is also compared to the human ADP ribosylation factor (ARF3) M33384, showing strong similarity (76% identity at the nucleic acid level and 87% identity at the amino acid level). The amino acid alignments of 740 AT # 120 to human ADP-ribosylation factor (ARF3) P16587 are shown in Figure 17 (SEQ ID NOs: 55-57) showing 87% identity and 90% positivity.

The high homology of nucleic acid and amino acid sequences between the two makes humanized 740 AT # 120 practical. The humanized sequence 740 AT # 120 H nucleic acid sequence is prepared by altering the 740 AT # 120 nucleic acid sequence to encode the same amino acid sequence encoding human M33384. The nucleic acid is altered by standard methods such as site-directed mutagenesis or DNA synthesis. Figure 18 (SEQ ID NOs: 58 and 59 for the nucleic acid sequence and SEQ ID NO: 60 for the amino acid sequence) shows the sequence alignment of 740 AT # 120 H to human ARF3 M33384.

Isolation of Arabidopsis thaliana ARF genomic clones

Genomic clones encoding Arabidopsis thaliana ARF can be isolated by probing a filter containing an Arabidopsis thaliana or BAC clone using a ³² P-labeled 740 AT # 120 NotI insert. Other members of the Arabidopsis thaliana ARF multigene family were identified using the program of the Genetics Computer Group of the University of Wisconsin. The BAC clone T08I13 located on chromosome II has a high degree of homology (78-86% identity at the nucleic acid level) with 740 AT # 120.

Isolation and Identification of cDNA Encoding Nicotiana bentamiana ARF

The 488 bp cDNA of the Nicotiana benthamiana cDNA library was amplified by PCR using the following oligonucleotides: ATARFK15, 5'-AAG AAG GAG ATG CGA ATT CTG ATG GT-3 '(upstream) (SEQ ID NO: 61), ATARFN176, 5'- ATG TTG TTG (PCR) using GAG AGC CAG TCC AGA CC-3 '(downstream) (SEQ ID NO: 62). In the reaction, the vent polymerase was inactivated with phenol / chloroform and the PCR product was cloned directly into the HincII site in Bluescript KS + (Stratagene). A plasmid map of KS + Nb ARF # 3, including the nicotienabantamidine ARF ORF in pBluescript KS +, is shown in FIG. The nucleotide sequence of Nicotiana benthamiana or KS + Nb ARF # 3, containing the partial ADP-ribosylation factor ORF, was determined by dideoxynucleotide sequencing. The nucleic acid sequence of KS + Nb ARF # 3 had strong similarity to other plant ADP-ribosylation factor sequences (82-87% identity at the nucleic acid level). Nucleotide sequence comparisons of Nicotiana benthamiana KS + Nb ARF # 3 and Arabidopsis thaliana 740 AT # 120 show high homology between them (Figure 20, SEQ ID NOs 63 and 64 respectively). The nucleic acid sequence of KS + Nb ARF # 3 shows a high degree of homology (77-87% identity and affinity) with plant, yeast and mammalian DNA encoding ARF (Table 7). Again, the high homology of the DNA encoding the GTP-binding proteins of yeast, plants, humans, cows and mice can transfuse the DNA of one donor organism into another host organism and effectively silence the endogenous genes of the host organism Lt; / RTI >

Full-length cDNA coding for ARF is isolated by screening a Nicotiana benthamiana cDNA library by colony hybridization using a ³² P-labeled Nicotiana benthamiana or KS + Nb ARF # 3 probe. Hybridization is performed at 42 ° C for 48 hours in 50% formamide, 5XSSC, 0.02M phosphate buffer, 5X Denhardt's solution, and 0.1mg / ml sheared female calf thymus DNA. The filters are washed at 65 ° C in 0.1X SSC, 0.1% SDS prior to radiography.

Rapid Separation of cDNA Encoding ARF, GTP Binding Proteins in Rice, Barley, Corn, Soybean, and Other Plants

Libraries containing full-length cDNAs from rice, barley, corn, soy, and other important crops can be obtained from shared and personal sources or prepared from plant mRNA. The cDNA is inserted into a small subcloning vector such as a viral vector or pBluescript (Stratagene), pUC18, M13, or pBR322. The transformed bacterium (E. coli) is then plated onto a large petri plate or bioassay plate containing the appropriate medium and antibiotic. Individual clones were selected using a robotic colony picker and arrayed in a 96 well microtiter plate. The cultures are incubated at 37 [deg.] C until the transformed cells reach the extinction period. Aliquots are taken and glycerol stocks are prepared for long term storage at -80 ° C. The remainder of the culture is used to inoculate additional 96 well microtiter plates containing selective medium and incubated overnight. DNA is isolated from the culture and stored at -20 ° C. Using a robotic unit such as Qbot (Genetix), the E. coli transformant or DNA is rearranged to a nylon filter or glass slide at a high density. Full-length cDNA encoding ARF from rice, barley, corn, soy, and other important crops was cloned into a ³² P-labeled or fluorescent Nicotiana benthamiana or KS + / Nb ARF # 3 probe, or Arabidopsis 740 AT # 120 NotI insert By screening various filters or slides by hybridization.

Rapid separation of genomic clones encoding ARF, GTP binding proteins in rice, barley, corn, soy, and other plants

Genomic libraries containing sequences from rice, barley, maize, soy and other important crops are obtained from shared and personal sources or are prepared from plant genomic DNA. BAC clones containing the entire plant genome are constructed and organized in order of least redundancy. Individual BACs are sheared into 500-1000 bp fragments and cloned directly into viral vectors. Approximately 200-500 clones, which completely encompass the entire BAC, will form the BAC virus vector sub-library. These libraries can be stored as bacterial glycerol stocks at -80 ° C and as DNA at -20 ° C. Genomic clones are identified by first probing the BAC filter using a ³² P-labeled or fluorescent Nicotiana benthamiana or KS + / Nb ARF # 3 probe, or a ³² P-labeled Arabidopsis 740 AT # 120 NotI insert. BACs that hybridize to the probe are selected and their corresponding BAC viral vector sub-libraries are used to produce infectious RNA. Plants transfected with the BAC sub-library are screened for loss of function (e. G., Growth inhibited plants). The inserts of these clones or their corresponding plasmid DNAs are identified by dideoxy sequencing. This provides a rapid method for obtaining genomic sequences for plant ARF or GTP binding proteins.

Rapid separation of cDNA encoding human ADP-ribosylation factor

Libraries containing full-length cDNAs from living organisms such as the brain, liver, heart, lung, etc. are obtained from a shared and personal source or prepared from human mRNA. The cDNA is inserted into a small subcloning vector such as a viral vector or pBluescript (Stratagene), pUC18, M13, or pBR322. The transformed bacterium (E. coli) is then plated on a large Petri plate or biopsy plate containing the appropriate medium and antibiotics. Individual clones are selected using a robotic colony picker and arranged in a 96 well microtiter plate. The cultures are incubated at 37 [deg.] C until the transformed cells reach the extinction period. Aliquots are taken and glycerol stocks are prepared for long term storage at -80 ° C. The remainder of the culture is used to inoculate additional 96 well microtiter plates containing selective medium and incubated overnight. DNA is isolated from the culture and stored at -20 ° C. Using a robotic unit such as Qbot (Genetix), the E. coli transformant or DNA is rearranged to a nylon or nitrocellulose filter or glass slide at high density. Length cDNA encoding ARFs derived from human brain, liver, heart, lung, etc. was hybridized using a ³² P-labeled or fluorescent Nicotiana benthamiana or KS + / Nb ARF # 3 probe or Arabidopsis 740 AT # 120 NotI insert By screening various filters or slides.

Construction of viral vectors containing human cDNA

ARF5 clone containing a nucleic acid insert of human brain cDNA library (Bobak et al., Proc. Natl. Acad. Sci. USA 86: 6101-6105 (1989)) was amplified by polymerase chain reaction (PCR) using the following oligonucleotides: HARFMIA, 5'-TAC CTA GGG CAA TAT CTT TGG AAA CCT TCT CAA G-3 '(upstream) (SEQ ID NO: 65), HARFK181X, 5'- CGC TCG AGT CAC TTC TTG TTT TTG AGC TGA TTG GCC AG- ) (SEQ ID NO: 66). The vent polymerase in the reaction is inactivated using phenol / chloroform. The PCR product is cloned directly into the XhoI and AvrII sites of TTO1A.

Example 12

Using a tobruvirus vector, the silencing of phytoene-unsaturated enzymes in Nicotiana benthamiana

Tobacco rattletombirus (TRV) is a bispecific sense, single-stranded RNA virus. TRV can be used in a variety of forms including Arabidopsis thaliana (unpublished data), Nicotiana species, Brassica campestris, Capsicum annuum, Chenopodium amaranticolor, Glycine max, Lycopersicon esculentum, Narcissus pseudonarcissus, Petunia X hybrida, Pisum sativum, Solanum tuberosum, Spirometer sp. It can infect a wide range of host plants, including Spinacia oleracea, Vicia faba (http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/) 72010004.htm # SymptHost). TRV RNA-1 encodes a protein associated with viral replication (replication enzyme, 134/194 kDa) as well as cysteine-rich protein (CRP 16 kDa) and migration (29 kDa of transfer protein (mp)) (Fig. PLSB-1, an improved mutant of TRV RNA-1, was isolated from the passage extract of PpK20 from other Nicotiana benthamiana plants (MacFarlane and Popovich. Efficient expression of foreign proteins in roots from tobravirus vectors. Virology, 267, 29-35 (2000)). ≪ / RTI > Plants inoculated with pLSB-1 RNA-1 exhibit gene silencing more extensively than plants inoculated with PpK20 RNA-1. Virion was purified from leaf tissues by PEG precipitation method (Gooding GV Jr, Hebert TT (1967) A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57 (11): 1285) (Qiagen ), Followed by cDNA synthesis using the cDNA synthesis system (Gibco BRL ) Using oligonucleotides 5'-TTAATTAAGCATGCGGATCCCGTACGGGCGTAATAACGCTTACGTAGGCGAGGGGTTTTAC-3 '. The full length TRV RNA-1 was PCR amplified using the oligonucleotides 5'-ATGAAGAGCATGCTAATACGACTCACTATAGATAAAACATTTCAATCCTTTGAACGC-3 '(upstream) and 5'-TTCATCTGGATCCCGGGCGTAATAACGCTTACGTAGGCG-3' (downstream) and cloned into pUC18 at the SphI / BamHI site. This TRV RNA-1 construct, pLSB-1, was identified by dideoxy nucleic acid sequencing and found to have a 29 point mutation compared to the published sequence for PpK20 RNA-1 (Visser, PB and Bol, JF 1999) ACCESSION AF 166084). All these point mutations are present in the replication enzyme gene, many of which encode amino acid substitutions. The mutant TRV RNA-1 viral sequences contained in pLSB-1 are as follows. 5'-

RNA-2 encodes a capsid protein and two unstructured proteins, 2b and 2c (Fig. 21.A). The TRV RNA-2 construct expressing GFP was derived from a full length clone of RNA2 of TRV isolate, PpK20 (Mueller et al., 1997. Journal of General Virology, 78, 2085-2088 (1997), MacFarlane and Popovich. Efficient expression of foreign proteins in roots from tobravirus vectors. Virology, 267, 29-35 (2000)). This TRV-GFP construct has the 2c gene of TRV RNA-2 replaced by a pea-early-browning virus (PEBV) coat protein promoter linked to GFP (MacFarlane and Popovich, 2000). The TRV-GFP construct was further modified by replacing the GFP gene with the PstI and NotI cloning sites to produce the plasmid pK20-2b-P / N. (PDS) gene was PCR amplified from the plasmid pWPF187 using the following oligonucleotides 5'-TGGTTCTGCAGTTATGCATGCCCCAAATTGGACTTG-3 '(upstream) and 5'-TTTTCCTTTTGCGGCCGCTAAACTACGCTTGCTTCTG-3' (downstream). This PCR product was then subcloned into pK20-2b-P / N in the positive direction. The resulting construct, TRV-PDS (Figure 21.B), was linearized with SmaI and transcribed using T7 RNA polymerase (Ambion mMessage mMachine). Transcript RNA2 was mixed with a transcript of the full-length clone of TRV RNA-1 (pLSB-1).

TRV-PDS was inoculated on Nicotiana benthamianaa. After 6-7 days, the yellowish white area began to appear on the leaves at the top. After 8-10 days, this yellowing area began to appear as a white area. Samples of TRV-PDS infected plants were analyzed using HPLC. HPLC analysis showed a significantly increased level of phytoene in the TRV-PDS infected plants when compared to the uninoculated control.

Example 13

Identification of nucleic acid sequences involved in the regulation of plant development by cytoplasmic inhibition of gene expression in the antisense orientation using virus-induced RNA (G-protein associated receptor)

This example again demonstrates that episomal RNA viral vectors can be used to deliberately manipulate signaling pathways in plants. In addition, our results suggest that the Arabidopsis antisense transcript may block the expression of nicotianabentamia gene.

A partial Arabidopsis thaliana cDNA library was placed under the transcriptional control of the tovomovirus subgenomic promoter in the RNA viral vector. Colonies of the transformed Escherichia coli were automatically taken using a Flexys robot and transferred to a 96 well flat bottom block containing 50 μg / ml of terrific broth (TB) Amp. Approximately 2000 plasmid DNAs were separated from the overnight culture using BioRobot and infectious RNA from 430 independent clones was directly applied to the plants. At 1-2 weeks after inoculation, the transfected Nicotiana benthamiana plants were visually monitored for changes in growth rate, morphology, and color. A set of plants transfected with 740 AT # 88 (Figure 22) showed a white phenotype on infected leaf tissue. DNA sequencing revealed that this clone contains the Arabidopsis G-protein-linked receptor open reading frame (ORF) in the antisense orientation.

DNA sequencing and computer analysis

The 758 bp NotI fragment of 740 AT # 88 containing the G-protein associated receptor cDNA was identified. The nucleotide sequence determination of 740 AT # 88 was performed by dideoxy termination using a double stranded template. Nucleic acid sequence analysis and amino acid sequence comparison were performed using the DNA Strider, PCGENE and NCBI Blast programs. Figure 23 shows the partial nucleic acid sequence (SEQ ID NO: 69) and amino acid sequence (SEQ ID NO: 70) of the 740 AT # 88 insert. The nucleotide sequence of 740 AT # 88 was compared with Brassica rapa cDNA L35812 (Fig. 24, SEQ ID NOs: 71 and 72), 91% identical and positive; Was 68% identical and positive to the octopus rhodopsin cDNA X07797 (Figure 25, SEQ ID NOs: 73 and 74). The homology of the DNA encoding rhodopsin from plants and animals with rhodopsin indicates that one system of genes can inhibit the expression of genes of other systems. The amino acid sequence derived from 740 AT # 88 was compared with Octopus rhodopsin P31356 (FIG. 26, SEQ ID NO: 75-77) and was 65% identical and positive. ≪ tb > TABLE 8 < tb > A comparison of amino acid sequence with D. discoideum and octopus rhodopsin is shown: 58-62% identity and 63-65% positive.

Example 14

Identification of nucleic acid sequences including Arabidopsis S18 ribosomal protein open reading frames

At 1-2 weeks after inoculation, the transfected Nicotiana benthamiana plants were visually monitored for changes in growth rate, morphology, and color. One set of plants transfected with 740 AT # 377 (Figure 27) was severely inhibited from growing. DNA sequence analysis (Figure 28, SEQ ID NO: 78) revealed that this clone contained the Arabidopsis S18 ribosomal protein open reading frame (ORF) in the antisense orientation.

Example 15

Identification of L19 ribosomal protein genes involved in the regulation of plant growth by cytoplasmic inhibition of gene expression using virus-induced RNAs

At 1-2 weeks after inoculation, the transfected Nicotiana benthamiana plants were visually monitored for changes in growth rate, morphology, and color. One set of plants transfected with 740 AT # 2483 (Figure 29) was severely inhibited from growing. DNA sequencing (Figure 30, SEQ ID NO: 79) revealed that this clone contained the Arabidopsis L19 ribosomal protein open reading frame (ORF) in the antisense orientation. The 740 AT # 2483 nucleic acid sequence showed a high degree of homology (77-78% identity and positivity) to the plant, L19 ribosomal protein gene (Table 9). In addition, the 740 AT # 2483 nucleic acid sequence showed a high degree of homology (71-79% identity and positivity) to yeast, insect and human L19 ribosomal protein genes (Table 9). Comparison of the amino acid sequence of 740 AT # 2483 with human, insect and yeast ribosomal protein L19 shows 38-88% identity and 61-88% positivity (Table 10). The high homology of the DNA encoding human, plant, yeast and insect-derived ribosomal L19 protein indicates that the gene of one organism can inhibit the gene expression of an organism from another system.

DNA sequencing and computer analysis

The NotI fragment of 740 AT # 909 containing the ribosomal protein L19 cDNA was identified. The nucleotide sequence determination of 740 AT # 909 (Figure 31) was performed by dideoxy termination using double stranded templates. Nucleic acid sequence analysis and amino acid sequence comparison were performed using the DNA Strider, PCGENE and NCBI Blast programs. Figure 32 shows nucleic acid alignment (sequence numbers 80 and 81, respectively) of 740 AT # 909 to the human S56985 ribosomal protein L19 cDNA. Figure 33 (SEQ ID NO: 82-84) shows amino acid alignment of 740 AT # 909 to human P14118 60S ribosomal protein L19. Table 11 shows the 740 AT # 909 nucleic acid sequence comparison for plants, fruit flies, yeast, and humans: 63-79% identity and positive. Table 12 shows a comparison of 740 AT # 909 amino acid sequences for plant, human, mouse, yeast, and insect L19 ribosomal proteins: 65-88% identity and 80-92% positive.

Example 16

Construction of cytoplasmic inhibition vector with positive sense containing Arabidopsis thaliana HAT7 homeobox-leucine zipper nucleic acid sequence

The Arabidopsis thaliana CD4-13 cDNA library was digested with Not I. DNA fragments of 500 to 1000 bp were separated by trough extraction and subcloned into the NotI site of pBS740. Escherichia coli C600 water-soluble cells were transformed with the pBS740 AT library and colonies containing Arabidopsis cDNA sequences were selected on 50 μg / ml of LB Amp.

HAT7 Homebone - Segregation of genes encoding leucine zipper

At 1-2 weeks after inoculation, the transfected Nicotiana benthamiana plants were visually monitored for changes in growth rate, morphology, and color. Plants transfected with 740 AT # 855 (Figure 34) were adequately inhibited from growing. Plasmid 740 AT # 855 contains the portions of TMV-U1 126, 193, and 30 kDa ORF, the TMV-U5 coat protein gene (U5 cp), the T7 promoter, the Arabidopsis thaliana CD4-13 NotI fragment, and the pUC19 plasmid. The TMV-U1 subgenomic promoter located within the negative strand of the 30 kDa ORF regulates the synthesis of CD4-13 subgenomic RNA.

DNA sequencing and computer analysis

740 AT # 855 was identified. Nucleic acid sequence analysis and amino acid sequence comparison were performed using the DNA Strider, PCGENE and NCBI Blast programs. 740 AT # 855 contained the Arabidopsis thaliana HAT7 homeobox-leucine zipper cDNA sequence. 740 AT # 855 and Arabidopsis thaliana HAT7 Homo box protein ORF (UO9340). Figure 36 (SEQ ID NO: 85-87) shows the nucleic acid sequence of 740 AT # 855 and Arabidopsis thaliana HAT7 homeobox protein ORF, and the amino acid sequence of Arabidopsis thaliana HAT7 homeobox protein ORF. 855 inhibits the expression of the HAT7 humobox protein in transfected Nicotiana bentamiana, including the 3'-untranslated region (UTR) of the Arabidopsis thaliana or HAT7 humobox protein ORF in the positive direction . Table 13 shows the nucleic acid sequence comparison between 740 AT # 855 and Arabidopsis thaliana, rat and human: 65-98% identity and positive.

Example 17

Identification of human nucleic acid sequences involved in the regulation of plant growth by cytoplasmic inhibition of gene expression using viral-derived RNA containing human nucleic acid sequences

Human brain cDNA libraries are obtained from shared and personal sources or prepared from human mRNA. The cDNA is inserted into a viral vector or a small subcloning vector, the cDNA insert is separated from the cloning vector with the appropriate enzyme, modified, and the NotI linker is attached to the cDNA blunt end. The human cDNA insert is subcloned into the NotI site of pBS740. Escherichia coli C600 water-soluble cells are transformed with the pBS740 sub-library, and colonies containing the human cDNA sequence are selected on 50 mu g / ml of LB Amp. DNA containing the viral human brain cDNA library is purified from transformed colonies and used to produce infectious RNA directly applied to plants. At 1-3 weeks after transfection, plants expressing the severe growth retardation phenotype are identified and their corresponding viral vector inserts are identified by nucleic acid sequencing.

Identification of human nucleic acid sequences involved in the growth control of host organisms by inhibition of the inherent gene expression using virus-derived RNAs containing human nucleic acid sequences

Human brain cDNA libraries are obtained from shared and personal sources or prepared from human mRNA. The cDNA is inserted into a viral vector or small subcloning vector, the cDNA insert is separated from the cloning vector with the appropriate enzyme, modified, and the NotI linker is attached to the cDNA blunt end. The human cDNA insert is subcloned into the NotI site of pFastBacl. Human cDNA inserts were removed from the shuttle plasmid pFastBac-HcDNA containing human cDNA inserts in pFastBacMaml as an EcoRI-XbaI fragment according to Condreay et al. (Proc. Natl. Acad. Sci. USA, 96: 127-132 -H cDNA was constructed. Recombinant viruses are generated using the Bac-to-Bac system (Life Technologies). Virus is further propagated by propagation in Spodoptera frugiperda cells. Phenotypic changes such as rate, shape, size, kinase activity, cytokine dissociation, response to excipients (eg, toxic compounds, pathogens, etc.), cell culture cleavage, serum free growth, gene activation, , Macroscopically, or by biochemical methods. Cells with phenotypic or biochemical changes are detected and subsequent sequencing of the cDNA clone or nucleic acid insert in the vector leading to the change is sequenced.

Humanization of plant analogs for the expression of plant-derived human proteins

To obtain the corresponding plant cDNA, human clones involved in causing changes in the transfected plant phenotype (e. G., Growth inhibition) are used as probes. The full-length plant cDNA is separated by hybridizing filters or slides containing the Nicotiana bentamiana cDNA using a ³² P-labeled or fluorescent human cDNA insert probe. Positive plant clones are identified by nucleic acid sequencing and compared with their human homologues. Plant codons encoding different amino acids are changed to codons that encode the same amino acids as their human homologues by site-directed mutagenesis. The resulting " humanized " plant cDNA encodes the same protein as the human clone. &Quot; Humanized " plant clones are used for the production of human proteins in transgenic or transformed plants by standard techniques. Because " humanized " cDNA is plant-derived, it is suitable for expression in plants.

Example 18

Gene silencing / simultaneous repression of genes induced by the transfer of RNA capable of forming its own base pairs to form double-stranded regions

The gene silencing was used to down-regulate gene expression in transgenic plants. Recent experimental evidence suggests that double-stranded RNA may be an effective stimulus for gene silencing / co-suppression in transformed plants. For example, Waterhouse et al. (Proc. Natl. Acad. Sci. USA 95: 13959-13964 (1998), incorporated herein by reference) discloses that virus resistance and gene silencing in plants are caused by simultaneous expression of sense and antisense RNA Lt; / RTI > Genetic silencing / simultaneous repression of plant genes can be induced by delivering RNA that can form its own base pairs to form double stranded regions.

This example shows a novel method for generating (1) RNA viral vectors capable of producing RNA capable of forming double stranded regions, and (2) silencing plant genes by using such viral vectors.

Step 1: Construction of a DNA sequence that generates an RNA molecule capable of forming its own base pair after being transcribed. Two identical, or nearly identical, double-stranded DNA sequences are ligated together in the reverse direction (i.e., head-tail or tail-head direction) with respect to each other, with or without a linking nucleic acid sequence between homologous sequences. The resulting DNA sequence is then cloned into a cDNA copy of the plant virus vector genome.

Step 2: Cloning, screening, and transcription of clones of interest using methods known in the art.

Step 3: Infect plant cells with transcripts of clones.

When the virus expresses a foreign gene sequence, the RNA of the foreign gene forms its own base pair to form a double-stranded RNA region. This approach is used as an arbitrary plant or non-plant gene and is used to silence a homologous plant gene to aid in the identification of the function of a particular gene sequence.

Although the present invention is described with respect to currently preferred embodiments, it should be understood that various modifications may be made without departing from the spirit of the invention.

All publications, patents, patent applications, and Web sites are hereby incorporated by reference to the same extent as if each individual publication, patent, patent application, or Web site was specifically and individually indicated to be incorporated by reference in its entirety.

Claims (29)

A method of associating a nucleic acid sequence of a donor organism with its function, comprising the steps of: (a) introducing a library of cDNA, genomic DNA or RNA inserts derived from a donor organism into a plant viral vector in a positive sense direction; (b) expressing each insert of said library in a host plant; (c) detecting the phenotype or biochemical change of the host plant due to expression of the insert of the library; (d) determining the sequence of the insert; And (e) associating the sequence of the insert with the phenotype or biochemical change. A method of associating a sequence with a function, comprising the steps of: (a) introducing into a plant viral vector a library of cDNA, genomic DNA or RNA inserts from a donor organism in the antisense orientation; (b) expressing each insert of said library in a host plant; (c) detecting the phenotype or biochemical change of the host plant due to expression of the insert of the library; (d) determining the sequence of the insert; And (e) associating the sequence of the insert with the phenotype or biochemical change. 3. The method according to claim 1 or 2, wherein said donor organism is selected from the group consisting of Monera, Protoctista, Fungi, and Animalia. The method according to claim 1 or 2, wherein the donor organism is human. 3. The method according to claim 1 or 2, wherein the donor organism is mouse. 3. The method according to claim 1 or 2, wherein the donor organism is a fruit fly. 7. The method according to any one of claims 4 to 6, wherein the library is derived from a tumor cell. 7. The method according to any one of claims 4 to 6, wherein the library is derived from an EST. The method according to claim 1 or 2, wherein the donor organism is a plant, and the donor plant and the host plant belong to different trees, trees, rivers, or gates. 3. The method according to claim 1 or 2, wherein the donor plant is selected from the group consisting of edible crops, seed crops, oil crops, ornamental crops and forestry crops. 3. The method according to claim 1 or 2, wherein said host plant is selected from the group consisting of edible crops, seed crops, oil crops, ornamental crops and forestry crops. 3. The method according to claim 1 or 2, wherein said host plant is nicotiana. 13. The method of claim 12, wherein the host plant is Nicotiana bentamiana or Nicotiana Cleverlandia. 3. The method according to claim 1 or 2, wherein the host plant is a terminal leaf. 3. The method according to claim 1 or 2, wherein the plant viral vector is derived from a single strand, positive sense RNA virus. 16. The method according to claim 15, wherein the plant virus vector is derived from the group consisting of a potivirus, a tobaemovirus, a bromovirus, a gemini virus, a noridivirus, and a tobrovirus. 16. The method of claim 15, wherein the single strand, positive sense RNA virus is a polymorphic virus. 3. The method according to claim 1 or 2, wherein the plant virus vector contains a natural or non-natural subgenomic promoter. 3. The method of claim 1 or 2, wherein said insert encodes a protein selected from the group consisting of a ribosomal protein, a GTP binding protein, a tumor suppressor, and a G-protein associated receptor. 3. The method of claim 1 or 2, wherein the phenotype change comprises growth rate, shape or color change. 2. The method of claim 1, wherein the plant virus vector containing the insert causes cytosolic inhibition of gene expression. 4. The method of claim 1, wherein the plant virus vector containing the insert causes overexpression of the polypeptide product. 3. The method of claim 1 or 2, further comprising annotating each insert sequence with its associated phenotype or biochemical change. 3. The method according to claim 1 or 2, further comprising the steps of: (1) determining the nucleic acid sequence homology between human and plant sequences (2) altering the nucleic acid sequence of the plant sequence such that the altered plant sequence encodes the same amino acid sequence as the human sequence. 3. The method according to claim 1 or 2, further comprising the step of using the function of the sequence in step (d) when increasing the crop yield. 17. The method of claim 16, wherein the plant virus vector is derived from Tobacco Rattle Virus. 16. The method of claim 15, wherein the plant virus vector is derived from barley stripe mosaic virus. A nucleic acid product containing the sequence of step (d) of claim 1 or 2. A polypeptide product encoded by the sequence of step (d) of claim 1 or 2.