WO2018010459A1 - Isolated promoter safes6 not expressed in endosperm and application of promoter safes6 - Google Patents

Abstract

Provided are an isolated promoter SAFES6 not expressed in endosperm and an application of the promoter SAFES6. Specifically, provided are a promoter SAFES6 capable of transcribing a heterologous nucleic acid sequence in plant tissue rather than in endosperm, and methods for preparing and using the promoter. Also provided are an expression cassette containing the promoter, a recombinant expression vector, and a method for obtaining a corresponding transgenic plant.

Description

An isolated endosperm does not express promoter SAFES6 and its application

Related application

The present application claims the following priority: Chinese Invention Patent Application filed on Jul. 15, 2016, the application number: 201610560043.4, entitled "An isolated endosperm does not express the promoter SAFES6 and its application".

Technical field

The invention relates to the field of biotechnology and crop genetic engineering technology. In particular, the present invention relates to a rice endosperm non-expression promoter SAFES6 and uses thereof.

Background technique

One of the goals of crop genetic engineering is to cultivate plants with the characteristics or traits required for agriculture. Technological advances in genetic engineering have enabled researchers to obtain the desired gene of interest and transform plants to obtain transgenic plants with the desired traits and characteristics. In one aspect, genes can be promoted to be expressed in plant cells or plant tissues that do not normally express such genes to confer the desired phenotype on these plants. On the other hand, transcription of a gene or part of a gene in the antisense orientation can prevent or inhibit the expression of an endogenous gene to achieve the desired target. The use of a promoter to directly control the transcription of a gene of interest is the most economical and effective means of regulating the expression of a gene of interest, and is also a feasible pathway for controlling the expression of risk.

Since the birth of the first tobacco GM crop in the United States in 1983, GM crops have been planted worldwide since the end of the year, and more than 1,000 approved products are available. Most of the current genetic improvement of these crops is focused on increasing yield, shortening the breeding cycle, enhancing disease resistance, improving nutrient content of agricultural products, and increasing biodiversity. Many GM crops and their products have entered the food distribution market and entered the dining table of thousands of households. Rice is one of the world's major food crops, and more than one-third of the world's population is dominated by rice. China is a populous country. Rice plays a very important role in China's national economy. In order to improve rice yield, resistance and quality, genetic engineering technology can be used to introduce exogenous target genes into cultivated rice to create transgenic rice. This has opened up a new way to solve the contradiction between food shortage and population growth.

Throughout the research and development of transgenic rice, the improved traits of rice transgenic engineering mainly involve disease resistance, herbicide resistance, quality improvement and insect resistance.

In terms of disease resistance, it is mainly transferred to rice fungi, bacterial, and nematode viruses. After introducing the Xa21 gene into rice, Christou et al. significantly increased the resistance of rice to bacterial blight and rice blast. He Yingchun, etc. The tobacco chitinase gene was introduced into rice to obtain high resistance to sheath blight and rice blast transgenic plants. Sichuan Agricultural University introduced the disease-resistant and insect-resistant genes into the restorer line of hybrid rice, and obtained the transgenic double-resistant hybrid rice.

In the 1980s, Monsanto Company of the United States took the lead in carrying out research on herbicide antibacterial genes and development of resistant varieties with its broad-spectrum and high-efficiency herbicide Roundup (glyphosate). The gene used in rice herbicide resistance in China is mainly the bar gene encoding PAT.

In terms of quality improvement, proline-rich genes, methionine- and lysine-rich genes, octahydrotoxin synthase and its dehydrogenase genes, glycinin genes and high-lysine genes have been successfully The introduction of rice and the acquisition of transgenic plants are of great significance for the development and utilization of rice products.

The Bacillus thuringiensis insecticidal crystal protein gene (Bt gene), insect protein inhibitor (PI gene) and plant lectin gene isolated from Bacillus thuringiensis have been successfully applied to rice resistance improvement.

However, everything has two sides. When genetically modified crops bring surprises and gospel to human beings, they also bring doubts and risks. The biosafety problem that GM rice may bring has become the focus of attention of the international community.

From a microscopic point of view, the introduction of a promoter may also pose a safety hazard to transgenic plants. For example, insertion of a promoter into a recessive viral gene originally integrated into the plant genome may reactivate the virus; insertion of the promoter upstream of a gene encoding a toxin protein may enhance the synthesis of the toxin; when the transgenic plant is When eaten by animals or humans, the promoter may be inserted upstream of a consensus oncogene through the level of the gene, activating and causing cancer. On the macro food safety issue, the focus of attention is whether the transgenic and its products are toxic or cause allergic reactions when eaten. Second, when the transgenic product carries a selectable marker gene that encodes some of it for clinical or intestinal use. Whether antibiotic resistant substances are likely to be transferred to microorganisms, thereby enhancing the resistance of pathogenic microorganisms and causing antibiotic failure (Ebinuma et al., 2001; Hohn et al., 2001).

Rice is one of the main foods, and the rice is the endogenous milk. In the transgenic rice, the accumulation of exogenous proteins in the endosperm and the possible food safety risks have become important factors restricting the commercialization process. In response to this problem, the promoter strategy is used to induce the inducible, tissue-specific and time-dependent promoters, so that the foreign gene is expressed only in the non-edible part, ensuring that the edible part does not contain the product of the exogenous gene expression, reducing its The potential risks brought about by human health are an effective way to promote the commercialization of genetically modified rice. At present, promoters that have been found to express non-endosperm mainly include stem-specific promoters, mesophyll cell-specific promoters, root-specific promoters, and the like. Promoter RSs1, which is specifically expressed in the phloem, as found in shi et al; the Crop Improvement and Utilization Research Group led by Roger Thilmony of the US Department of Agriculture has recently discovered a tissue-specific promoter, LP2, which is a receptor in photosynthesis. The gene, which is highly active in the leaves of transgenic rice, is not detected in seeds and flowers; the rolD promoter of Agrobacterium rhizogenes found in Varvara et al. is expressed only in roots and transferred to tomato plants of interest. However, in domestic research, the invention and research of non-endosperm expression promoters have rarely been reported. It is only found in the non-endosperm expression promoter OsTSP I invented by Yang Jianbo and so on. It is only in rice non-endosperm tissues (roots, stems, leaves, etc.) In the expression, not expressed in the endosperm. And the promoter P _D540 and the like which are found only in the green tissue part of rice discovered by Meng Cai et al.

Genetic engineering can fundamentally change the structure of living organisms. This is a major advance in the history of science, and it may also bring some degree of hidden danger to human society. Although GM crops do have potential food safety and environmental safety issues, they must not be a reason to hinder their development. Instead, they should find problems and work hard to solve them. If GM rice cannot be finally commercialized for production, then the best research results have no application value. Therefore, using the promoter strategy, the foreign gene is expressed only in the non-edible part, ensuring that the edible part does not contain the product of the expression of the foreign gene, that is, vigorously carrying out the study of the functional genome, and excavating and separating the non-endosperm expression with practical value. It can reduce the potential risks of genetically modified rice to human health, eliminate the public's prejudice and concerns about genetically modified rice, give full play to the advantages of genetically modified breeding, and cultivate more and better new varieties. Thereby guiding the development of genetically modified crops in China in a healthy and orderly manner.

Summary of the invention

The present invention contemplates providing a promoter that is not expressed in rice endosperm, and the invention also provides an expression cassette, recombinant expression vector, transgenic plant or plant part comprising the promoter. In one embodiment disclosed herein, the promoter is linked to a heterologous transcribable polynucleotide sequence. Also provided herein are methods of making and using the promoters disclosed herein, including an expression cassette comprising a promoter, a recombinant expression vector, and a transgenic plant or plant part comprising a promoter linked to a heterologous transcribable polynucleotide sequence.

Thus, in one aspect, the invention provides an isolated endosperm non-expressing promoter SAFES6, characterized in that said endosperm non-expressing promoter SAFES6 comprises at least one of the following polynucleotide sequences:

(a) a polynucleotide sequence comprising the nucleotide sequence shown as SEQ ID NO: 1 in the Sequence Listing;

(b) a nucleotide sequence having at least 85% homology to the nucleotide sequence shown by SEQ ID NO: 1 in the Sequence Listing and having the same function;

(c) a nucleoside obtained by adding, substituting or deleting one or more nucleotides in the nucleotide sequence shown by SEQ ID NO: 1 in the Sequence Listing, and SEQ ID NO: 1 in the Sequence Listing a nucleoside with the same function as an acid sequence Acid sequence;

(d) a nucleotide sequence complementary to a nucleotide sequence comprising SEQ ID NO: 1 in the Sequence Listing,

Wherein the endosperm non-expressing promoter SAFES6 is capable of ligating to a heterologous transcribable polynucleotide sequence and is capable of directing transcription of the heterologous transcribable polynucleotide sequence in non-endosperm tissue.

In another aspect, provided herein is an expression cassette comprising the promoter and a heterologous transcribable polynucleotide sequence linked to the promoter. Wherein the promoter is capable of eliciting transcription and expression of the heterologous transcribable polynucleotide sequence in plant non-endosperm tissue transformed with the expression cassette. The heterologous transcribable polynucleotide sequence is a gene having a function of altering a crop trait. The heterologous transcribable polynucleotide sequence described therein is inserted into the expression cassette in a sense orientation. Alternatively, the heterologous transcribable polynucleotide sequence is inserted into the expression cassette in an antisense orientation. In certain embodiments, the transcribable polynucleotide sequence transcribes an insect resistant protein.

In another aspect, provided herein is a recombinant expression vector comprising the promoter or the expression cassette, the promoter being capable of being linked to a heterologous transcribable polynucleotide sequence. The heterologous transcribable polynucleotide sequence described therein is a gene having a function of altering a crop trait.

In another aspect, the present invention provides a method of improving plant growth characteristics without altering plant endosperm traits, the method comprising:

(1) obtaining the endosperm non-expression promoter SAFES6 according to claim 1;

(2) linking the endosperm non-expression promoter SAFES6 to a gene having a function of altering crop traits to obtain a corresponding recombinant vector;

(3) transferring the recombinant vector into a cell of a target plant;

(4) cultivating the corresponding transgenic plant using the cells;

(5) selecting, from the obtained transgenic plants, the gene having the function of changing crop traits expressed in the non-endosperm site, and the plant not expressing at the endosperm, the trait of the plant at the non-endosperm site is improved, and the endosperm site is not Affected.

In a further aspect, the invention provides a method of transforming a plant or plant part, the method comprising stably transforming the plant or plant part with the expression cassette or the recombinant expression vector. The plant part is selected from the group consisting of a cell, a protoplast, a cell tissue culture, a callus, a cell mass, a germ, a pollen, an ovule, a petal, a style, a stamen, a leaf, a root, and a root. Tips and anthers. The plant is selected from the group consisting of a food crop, a vegetable crop, a flower crop, and an energy crop. In a specific embodiment, the plant part is rice callus and the plant is rice.

In a further aspect, the invention provides a method of directing expression of a transcribable polynucleotide sequence in a plant cell, comprising ligating said promoter to a heterologous transcribable polynucleotide sequence to construct a recombinant expression vector, and The recombinant expression vector transforms the plant cells to produce transformed plant cells and regenerate the transgenic plants.

The following definitions and methods are provided to better define the invention and to guide the general practitioner in practicing the invention. Unless otherwise indicated, the terms should be understood according to the ordinary usage of one of ordinary skill in the relevant art.

The terms "DNA sequence", "nucleic acid sequence" and "nucleic acid molecule" refer to a double-stranded DNA molecule of genomic or synthetic origin.

The term "expression" refers to the transcription of a gene to produce a corresponding mRNA that is translated to produce the corresponding gene product (ie, a peptide, polypeptide or protein).

The term "expression cassette" refers to a DNA molecule comprising a transcribed DNA molecule linked to one or more regulatory elements.

The term "recombinant expression vector" refers to a class of specialized vectors designed and constructed to specifically regulate the expression of a foreign gene and enable efficient expression of the cloned gene in transformed host cells. Vectors typically include one or more expression cassettes. A DNA molecule comprising a combined DNA molecule that does not occur naturally without human intervention. The term "recombinant vector" refers to, for example, a plasmid, cosmid, virus, self-replicating sequence, phage or linear single-stranded, circular single-stranded, linear double-stranded or circular double-stranded DNA or RNA nucleotide sequence. The recombinant vector can be of any origin and can undergo genome integration or self-replication.

The term "expression of antisense RNA" refers to an RNA molecule that is inversely complementary to a native mRNA. It is produced by transcription of a nonsense strand in double-stranded DNA and can be used to prevent the translational activity of mRNA complementary thereto present in cells transformed thereby. The gene encoding the antisense RNA is used to block the translation of the mRNA of the gene of interest (ie, complementary to the antisense RNA) into protein expression.

The term "sequence homology" refers to the extent to which two optimal alignment DNA sequences are identical. For example, a reference sequence and another DNA sequence maximize the number of nucleotide matches in the sequence alignment. The term "reference sequence" as used herein refers to the DNA sequence of SEQ ID NO: 1.

The term "heterologous" and "exogenous" refers to the interrelationship between two or more nucleic acid or protein sequences of different origin. For example, a combination between a promoter and a coding sequence is not normally found in nature, and the two are heterologous to each other. Furthermore, a particular sequence may be foreign to the cell or organism into which it is inserted (ie, it is not normally found in a particular cell or organism under natural conditions).

The term "ligation" refers to a functional spatial arrangement of two or more nucleotide regions or nucleotide sequences. Such as The positional relationship of the mover region to the nucleic acid sequence allows the nucleic acid sequence to be regulated by the promoter region to direct transcription. At this point, the promoter region is "linked" to the nucleic acid sequence.

The term "transcribed polynucleotide sequence" refers to any DNA molecule capable of being transcribed to an RNA molecule, including, but not limited to, those having a protein coding sequence and those producing an RNA molecule having a sequence suitable for gene suppression. The DNA molecule type may include, but is not limited to, a DNA molecule from the same plant, a DNA molecule from another plant, a DNA molecule from a different organism, or a synthetic DNA molecule, such as a DNA molecule containing antisense information of the gene, Or a transgenic DNA molecule encoding an artificial, synthetic or otherwise modified version. Exemplary transcribable DNA molecules incorporated into the recombinant expression vectors of the present invention include the reporter gene GUS, a codon-optimized synthetic Bt (Bacillus thuringiensis Bacillus thuringiensis) protein-encoding gene mCry1Ab.

The term "promoter" or "promoter region" refers to a non-coding nucleotide sequence of a particular function located immediately upstream of the 5'-end of the gene, immediately adjacent to the transcription initiation site. RNA polymerase initiates transcription of a gene by binding to it. The promoter or promoter region provides RNA polymerase or other recognition site for transcription initiation factors. The strength or activity of the promoter can be measured by the amount of RNA produced by its transcription, or by the amount of protein accumulated in the cell or tissue, or by the transcriptional activity of other promoters compared thereto.

The term "transformation" refers to the process by which foreign DNA is introduced into a cell, ie, the introduction of the nucleic acid into a recipient host. The term "host" refers to bacterial cells, fungal, animal or animal cells, plants or seeds, or any plant part or tissue, including plant cells, protoplasts, callus, roots, tubers, seeds, stems, leaves, shoots, embryos. And pollen.

The term "transgenic plant" directs the incorporation of a nucleic acid into a plant whose genome is stably integrated. For example, integration into the nuclear genome.

The term "a gene having a function of altering a crop trait" refers to a transcribable DNA molecule that is expressed in a particular plant tissue, cell or cell type, imparting desired characteristics to the plant. Products of genes with functions that alter crop traits can function in plants to alter plant morphology and physiology. In one embodiment of the invention, the promoter of the invention is incorporated into a recombinant expression vector such that the promoter is ligated into a transcribable DNA molecule having a gene that alters the function of the crop trait. In a transgenic plant containing such a recombinant expression vector, expression of a gene having a function of altering a crop trait can confer a favorable trait to the plant. Advantageous traits may include, for example, but are not limited to, herbicide tolerance, insect resistance, disease resistance, stress resistance, improved plant growth and development, improved yield, improved protein content, improved fruit ripening, biopolymer production, improved flavor Hybrid seed breeding effect.

In the present invention, the inventors of the present application found a 1931 bp DNA sequence having transcriptional regulatory activity in the genome of Oryza sativa L cv. Nipponbare, which has a target gene in rice. The role of endosperm tissue expression. Further, the DNA sequence shown in SEQ ID No: 1 in the Sequence Listing was obtained by isolation and cloning. Named SAFES6 or the promoter SAFES6.

It should be noted that in the DNA sequence of the promoter of SEQ ID No: 1, the sequence "gcattcaaga aagcaatcag cc" at the beginning of the sequence is a retention sequence of the forward primer used in obtaining the promoter, for a total of 22 bp; the sequence at the end of the sequence " Ggcgaccgat cgattagcta gc" is the retention sequence of the reverse primer used in the promoter process (the retention sequence is complementary to the corresponding sequence of the reverse primer), for a total of 22 bp. It should be emphasized that the promoter mentioned herein may refer to the entire DNA sequence as described above, or to the DNA sequence after the above primer retention sequence is removed. Even if a person skilled in the art obtains a similar sequence using other primers on the basis of the present invention, it falls within the scope of the present invention.

The recombinant expression vector of the present invention is called pCAMBIA1391-SAFES6, and the recombinant expression vector is a recombinant expression vector obtained by constructing the sequence shown in SEQ ID No: 1 or SAFES6 or promoter SAFES6 in pCAMBIA1391, which is referred to herein as pCAMBIA1391-SAFES6. . The inventors ligated the promoter SAFES6 sequence to the crop binary expression vector pCAMBIA1391, and obtained the corresponding recombinant plasmid (ie, recombinant expression vector), and SAFES6 drives GUS gene expression. The recombinant plasmid was used to transform Agrobacterium tumefaciens strain EHA105, and then transformed into rice by Agrobacterium-mediated method to obtain transgenic rice plants. The histochemical examination of the obtained transgenic rice showed that the transgenic plants had strong GUS expression in roots, stems, sheaths, leaves and embryos, but not in endosperm. Thus, it was confirmed that the 1931 bp sequence has an activity of driving gene-specific expression in non-endosperm tissues.

Quantitative analysis of GUS gene showed that the expression of rice SAFES6 promoter in leaf and leaf sheath tissues was significantly stronger than that of rice constitutive promoter ACTIN promoter commonly used in genetic engineering; in root and stem tissues, the activity of two promoters was basically equivalent; In the tissue, the SAFES6 promoter activity was slightly weaker than the ACTIN promoter; whereas in the endosperm, the SAFES6 promoter was inactive.

In pCAMBIA1391-SAFES6, mCry1Ab (Bac protein thuringiensis, Bt protein encoding gene, independent design of Anhui Academy of Agricultural Sciences, ZL 2009 10185774.5) was used to replace the GUS gene, and the expression vector of Bt driven by SAFES6 was obtained, which was named SAFES6-BT. In the relevant transgenic lines, the Bt expression level of SAFES6-BT plants in leaf and root tissues was slightly stronger or similar than that of the ACTIN promoter-driven Bt, while in the endosperm, ACTIN-BT was expressed, while SAFES6 -BT is not detected (below the detection line).

Technical effect

The endosperm of the invention does not express the promoter SAFES6 can regulate the non-endosperm part of the gene in the plant Medium expression has significant value in practical applications. For example, the promoter regulates the expression of the target gene in green tissues and roots, but does not express in the endosperm, improves the growth performance of the rice, and reduces the food safety risk of the rice, and facilitates commercial promotion of the genetically modified rice.

Sequence table

SEQ ID NO: 1 represents the SAFES6 promoter.

SEQ ID NOS: 2-11 represent the primer sequences.

DRAWINGS

Figure 1 shows the SAFES6 promoter and its PCR amplification primer sequence, showing the SAFES6 promoter with PCR primers, 1943 base pairs, also showing HindIII sites: 1-6, EcoRI sites: 1938- 1943;

Figure 2 is a schematic representation of the construction of the SAFES6 promoter in the pCAMBIA1391 vector plasmid, in which A is a schematic representation of pCAMBIA1391 and B is a schematic representation of pCAMBIA1391-SAFES6, which shows the expression of the Gus gene downstream of the SAFES6 promoter.

Fig. 3 is a view showing the results of enzyme digestion verification of the promoter of the present invention.

Figure 4 is a schematic diagram showing the results of using the SAFES6 promoter to drive Gus gene expression. The figure shows the results of Gus staining in various parts of rice. The Gus gene is in root (a), stem (b), leaf sheath (c), leaf (d), Strong expression in embryo (e), not expressed in endosperm, scale in the figure = 2.5mm.

Figure 5 is a graph showing the results of quantitative analysis of GUS of pCAMBIA1391-SAFES6 and pCAMBIA1391-ACTIN plants.

Figure 6 is a schematic representation of the SAFES6-BT expression cassette. The expression cassette comprises two expression cassettes, namely an expression cassette consisting of a CaMV35S promoter, a hyg gene, a CaMV terminator, and an expression cassette consisting of a SAFES6 promoter, a mCry1Ab gene, and a NOS terminator. Among them, LB: left border; CaMV terminator: CaMV terminator, length 0.22kb; Hyg: hygromycin gene hygromycin, length 1.86Kb; CaMV35S promoter: CaMV35S promoter, length 0.53Kb; SAFES6promoter: SAFES6 promoter, length 1.96Kb mCry1Ab: Bacillus thuringiensis (Bt) protein-coding gene, length 1.84Kb; NOS terminator: NOS terminator, 0.25Kb long; RB: right border.

Figure 7 is a schematic diagram of the ACTIN-BT expression cassette. The expression cassette comprises two expression cassettes, namely an expression cassette consisting of a CaMV35S promoter, a hyg gene, a CaMV terminator, and an ACTIN initiation Expression cassette consisting of a mover, mCry1Ab gene, and NOS terminator. Among them, LB: left border; CaMV terminator: CaMV terminator, length 0.22kb; Hyg: hygromycin gene hygromycin, length 1.86Kb; CaMV35S promoter: CaMV35S promoter, length 0.53Kb; ACTIN promoter: ACTIN promoter, length 1.3 Kb; mCry1Ab: Bacillus thuringiensis (Bt) protein-coding gene, length 1.84Kb; NOS terminator: NOS terminator, 0.25Kb long; RB: right border.

detailed description

The following examples are provided to illustrate the invention, and the following examples are intended to illustrate and not to limit the invention. Those skilled in the art will appreciate that the techniques disclosed in the examples which follow represent techniques discovered by the inventors in the practice of the invention. However, it will be apparent to those skilled in the art that <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; The scope of the present invention is to be considered as illustrative and not restrictive.

The experimental methods in the following examples are conventional methods unless otherwise specified. The biochemical reagents, carrier consumables and the like used in the following examples are commercially available products unless otherwise specified.

Example 1. Acquisition of the SAFES6 promoter

Step 1. Design of primers

According to the whole genome sequence of the rice variety Oryza sativa L cv. Nipponbare provided in NCBI, the amplification primers were designed according to the sequence of the rice promoter SAFES6 (ie the sequence shown in SEQ ID No: 1 in the sequence listing), and according to the selected The characteristics of the vector and the target gene are designed to design the restriction sites of the primers. The seed of the rice variety Nipponbare used in the present invention was obtained from the seed bank of the Rice Research Institute of Anhui Academy of Agricultural Sciences.

In this example, the rice binary expression vector pCAMBIA1391 (part A in Figure 2, from CAMBIA, for public use of the vector, the Anhui Provincial Academy of Agricultural Sciences, Genetically Modified Organisms Product Supervision and Testing Center) is taken as an example. Because of the Gus gene, the specifically designed primers are: forward primer (SEQ ID No: 2) 5' end with restriction enzyme site HindIII (AAGCTT), reverse primer (SEQ ID No: 3) 5' end with enzyme cleavage site Point EcoRI (GAATTC), the primer sequence is as follows:

Forward primer: 5'-AAGCTTGCATTCAAGAAAGCAATCAGCC-3'

Reverse primer: 5'-GAATTCGGCGACCGATCGATTAGCTAGC-3'

Synthesized by Shenzhen Huada Gene Company.

Step 2. Acquisition of the promoter SAFES6

Using the rice variety Nipponbare DNA as a template, the forward primer and reverse primer were used to amplify the promoter SAFES6 according to the conventional PCR system (using the conventional PCR amplification method, the specific details of the amplification method can be referred to the literature: Bachmann H S, Siffert W, Frey U H. Successful amplification of extremely GC-rich promoter regions using a novel'slowdown PCR 'technique. Pharmacogenetics, 2003, 13(12): 759-66.) The following amplification procedure was employed:

The template DNA was pre-denatured for 5 min at 95 °C. Then enter the denaturation-annealing-extension cycle: denaturation at 95 °C for 30 s, the template DNA double strands are decomposed into single strands; annealing at 58 °C for 30 s, primers hybridize with DNA single-stranded template to form DNA template-primer complex; 72 °C extension for 2 min 30 s, DNA The template-primer complex was synthesized by Taq DNA polymerase using dNTP as the reaction material and the target sequence as a template. Based on the principle of base pairing and semi-reserved replication, a new strand complementary to the template DNA strand was synthesized. A total of 35 cycles. Finally, it was extended at 72 ° C for 10 min.

The PCR-amplified target fragment was recovered (see Figure 1, the sequence of the promoter, the 5'-end and the 3'-end cleavage site are shown in Figure 1), and ligated into the PGEM-T-Easy vector plasmid (purchased) The PGEM-T-Easy vector carrying the desired fragment was transformed into E. coli XL-Blue competent cells by heat shock method from Promega, mixed according to the ratio in the vector specification. The E. coli bacterial solution was uniformly coated on an LB plate medium, and cultured at 37 ° C for 24 hours. Using the forward and reverse primers of the cloning promoter, the obtained single E. coli cell clones were subjected to colony PCR verification, and the positive clones were screened out. Subsequently, two positive cell clones were picked and cultured overnight, and the cells were collected for extraction of the PGEM-T-Easy vector plasmid containing the promoter SAFES6 fragment. The resulting plasmid was subjected to double enzyme digestion verification using HindIII and EcoRI, as shown in FIG. Positive clones that were double-identified by colony PCR validation and restriction enzyme digestion were sent to Invitrogen for sequencing. The correct clone was verified to be the promoter SAFES6 to be obtained, and the nucleic acid sequence thereof is shown in SEQ ID No: 1.

Example 2, Construction of a Crop Expression Vector and Transformation of Agrobacterium

From the E. coli positive clone obtained in the above "obtainment of promoter SAFES6", the PGEM-T-Easy vector plasmid containing the promoter SAFES6 fragment was extracted and digested with HindIII and EcoRI. The promoter SAFES6 fragment was recovered. At the same time, the crop expression vector pCAMBIA1391 was digested with HindIII and EcoRI, and the pCAMBIA1391 fragment after digestion was recovered. The pCAMBIA1391 vector contains a promoter-free reporter gene Gus (the promoter to be studied in the present invention is inserted upstream of the Gus gene to drive the expression of the Gus gene, and the expression characteristics of the Gus gene reflect the nature of the promoter under study. The SAFES6 fragment and the pCAMBIA1391 fragment recovered above were ligated with T4 DNA ligase (purchased from TaKaRa) to obtain a crop expression vector pCAMBIA1391-SAFES6 in which the promoter SAFES6 was fused with the Gus gene (Fig. 2B). Referring to the freeze-thaw method proposed by Hofgen et al. (Hofgen R, Willmitzer L. Storage of competent cells for Agrobacterium transformation. 1988. Nucleic Acids Res 16:9877.), the crop expression vector was transferred to Agrobacterium tumefaciens EHA105 ( Rice Group of the Organized Supervision and Testing Center for Genetically Modified Organisms of the Ministry of Agriculture of Anhui Academy of Agricultural Sciences).

Example 3: Promoter SAFES6 drives expression of Gus reporter gene

Step 1: Agrobacterium-mediated transformation of rice

After the mature Nipponbare rice seeds are removed from the glumes, the seeds are soaked in 70% alcohol for 1 min, and the alcohol is poured off. The seeds were soaked for 40 min with a solution of 50% sodium hypochlorite (the effective chlorine concentration of the stock solution greater than 4%) containing 1 drop of Tween 20 and shaken evenly on a shaker (150 r/min). Sodium hypochlorite was poured off and washed with sterile water for 5 times until the solution was clear without sodium hypochlorite. Soak the seeds in sterile water overnight. The embryos were peeled off along the aleurone layer of the seeds with a scalpel, and the embryos were inoculated on callus induction medium. After dark culture at 30 ° C for 11 days, the callus was separated from the endosperm and the embryo, and the primary callus with good degerization and vigorous division was pre-cultured for 3 to 5 days for Agrobacterium transformation.

Agrobacterium-mediated genetic transformation using Agrobacterium tumefaciens transformed into a recombinant expression vector during the above-mentioned "construction of crop expression vector and transformation of Agrobacterium", the genetic transformation, transformation of transformants and regeneration of transgenic plants are referred to Yongbo Duan (Yongbo Duan, Chenguang Zhai, et al. An efficient and high-throughput protocol for Agrobacterium mediated transformation based on phosphomannose isomerase positive selection in Japonica rice (Oryza sativa L.) [J]. Plant Cell Report, 2012. DOI 10.1007/ S00299-012-1275-3.) and other proposed methods. The method is roughly divided into the following steps:

(1) Callus induction: The sterilized seeds were soaked in sterile water at 30 ° C overnight, and the embryos were peeled off on an induction medium with a scalpel. Each dish (100×25mm disposable plastic culture) The dish containing 50 mL of induction medium was uniformly placed in 12 embryos, and the callus was induced to stand for 2 to 3 weeks at 30 ° C in the dark to grow a pale yellow granular callus. (Induction medium components: N6majors, MS iron salts, B5minors, B5vitamins, 500mg/L proline, 500mg/L glutamine, 300mg/L casein enzymatic hydrolysate, 30g/L sucrose, 2mg/L 2,4-D, 3g/ L phytagel, pH 5.8)

(2) Pre-culture: The granular callus was selected from the induction medium and placed on a new induction medium, and cultured at 30 ° C for 3 to 5 days in the dark.

(3) Infection and co-culture: Transfer the pre-cultured callus to a 50mL sterile tube, add Agrobacterium liquid (OD600=0.2) for 20min, pour out the bacteria solution, and use a sterile filter paper to make the callus The residual bacterial solution is blotted dry. The callus was evenly spread on the co-cultivation medium and cultured in the dark at 23 ° C for 2 to 3 days. (The total medium composition is: N6majors, MS iron salts, B5minors, B5vitamins, 500mg/L proline, 500mg/L casein enzymatic hydrolysate, 30g/L sucrose, 2mg/L 2,4-D, 3g/L phytag-el, 100μL/L acetosyingone, 4g/L phytagel, pH 5.2)

(4) Recovery: The co-cultured callus was transferred to a recovery medium and cultured in the dark at 30 ° C for 3 to 5 days.

(Recovery medium composition: N6majors, MS iron salts, B5minors, B5vitamins, 500mg/L proline, 500mg/L glutamine, 500mg/L casein enzymatic hydrolysate, 30g/L sucrose, 2mg/L 2,4-D, 250mg/ L carbenicillin, 3g/L phytagel, pH 5.8)

(5) Screening: The embryogenic calli with bright yellowish granules without plaques were selected from the screening medium and inoculated on the screening medium, 30 capsules per dish. Incubate in the dark at 30 ° C for 2 to 3 weeks until a new resistant granular granule callus grows. (Screening medium composition: N6majors, MS iron salts, B5minors, B5vitamins, 500mg/L proline, 500mg/L glutamine, 500mg/L casein enzymatic hydrolysate, 10g/L sucrose, 20g/L Mannose, 2mg/L 2,4 -D, 3g/L phytagel, 250mg/L carbenicillin, 25mg/L hygromycin, pH 5.8)

(6) Differentiation: Each transformation event selects three independent embryogenic callus to a certain area of differentiation medium, and cultures in a 30°C light culture room (16h light/8h darkness) for 3 to 4 weeks, until the seedling grows. . (Differentiation medium components are: N6majors, MS iron salts, B5minors, B5vitamins, 1g/L casein enzymatic hydrolysate, 30g/L sucrose, 30g/L sorbitol, 500mg/L MES, 2.5mg/L CuSO ₄ , 0.5mg/L KT, AA amino acids, 2g/L phytagel, pH 5.8)

(7) Rooting: Two robust seedlings were selected from each region and transplanted to rooting medium. The tissue culture chamber was cultured in a photoperiod (16h light/8h dark) at 30°C for about three weeks, and identified and transplanted to the field. (The rooting medium composition is: 1/2MS basal salts (2.165g/L), B5vitamins, 1.0g/L casein enzymatic hydrolysate, 20g/L sucrose, 0.2mg/L NAA, 125mg/L carbenicillin, 3.5g/L phytagel , 25mg/L hygromycin, pH5.8)

The rice plant containing the pCAMBIA1391-SAFES6 expression vector was finally obtained, which we call the pCAMBIA1391-SAFES6 plant.

Step 2. GUS histochemical staining

Agrobacterium transformation was carried out according to the method proposed by Jefferson (Jefferson RA et al. GUS fusion: β-Glucuronidase as a sensitive and versatile gene fusion marker in higher plant [J]. EMBO J., 1987, 6: 3901-3907) The posterior tissue was subjected to GUS histochemical staining. The tissue to be stained was evacuated, then immersed in the staining solution, and stained at 37 ° C for 24 hours. Then decolorize, decolorize the tissue soaked in 95% ethanol, until the green tissue material is white, the staining results are shown in Figure 4, from the figure can be seen GUS in the root (a), stem (b), leaf sheath (c) Strong expression in leaves (d) and embryos (e), not expressed in endosperm (e).

Example 4 Quantitative analysis of promoter SAFES6 activity

The pCAMBIA1391-SAFES6 plants and the control pCAMBIA1391-ACTIN plants (pCAMBIA1391-ACTIN plants were obtained by directly planting the corresponding seeds, and the seeds containing pCAMBIA1391-ACTIN were from Cornell University, USA, and are currently used in genetic engineering. The constitutive promoter rice ACTIN-driven Gus gene, preserved by the Ministry of Agriculture, Anhui Provincial Academy of Agricultural Sciences, GMO biochemical component inspection and testing center) leaf, sheath, stem, root, flower, endosperm sample, using the total plant RNA of Tiangen Biochemical Technology Co., Ltd. The extraction kit extracts the total RNA in each sample, and then the total RNA extracted is reverse-transcribed into cDNA using the FastQuant RT kit of Tiangen Biotechnology Co., Ltd., and the cDNA obtained from each sample is used as a template, respectively The ACTIN gene is an internal reference gene, and the SuperReal PreMix Plus real-time fluorescent quantitative PCR premix is used as a reagent in the ABI company's PRISM 7500 fluorescence PCR instrument. The pCAMBIA1391-SAFES6 and pCAMBIA1391- are detected by qRT-PCR reaction. Interview of various tissue parts in ACTIN transgenic plants The expression intensity of the gene Gus, which reflects the corresponding promoter activity. Among them, the quantitative qRT-PCR primer for calibrating the internal reference gene ACTIN is (SEQ ID No: 4-5):

ACTIN upstream primer: 5'-CCTTCAACACCCCTGCTATG-3’

ACTIN downstream primer: 5'-CAATGCCAGGGAACATAGTG-3’

The qRT-PCR primers used to detect Gus gene expression are (SEQ ID No: 6-7):

Gus upstream primer: 5'-TACGGCAAAGTGTGGGTCAATAATCA-3’

Gus downstream primer: 5'-CAGGTGTTCGGCGTGGTGTAGAG-3’

The results of quantitative analysis showed that, as shown in Fig. 5, it can be seen from the figure that the expression of rice SAFES6 promoter in leaf and leaf sheath tissues is significantly stronger than that of rice constitutive promoter ACTIN promoter commonly used in genetic engineering; In the tissue, the activity of the two promoters is basically equivalent; in the flower tissue, the rice SAFES6 promoter is slightly weaker than the ACTIN promoter; while in the endosperm, the rice SAFES6 promoter is inactive.

Example 5: Activity analysis of promoter BT driven by SAFES6

1. Construction of SAFES6-BT and ACTIN-BT expression vectors

According to the vectors pCAMBIA1391-SAFES6, pCAMBIA1391-ACTIN and gene mCry1Ab (Bacillus thuringiensis (Bt) protein-coding gene, independently designed by Anhui Academy of Agricultural Sciences, published in patent ZL 200910185774.5), the primers with restriction sites were designed, and the primers were forward. The 5' end carries the restriction enzyme site EcoRI (GAATTC), and the reverse primer 5' end carries the restriction enzyme site SpeI (ACTAGT). The primer sequence is as follows (SEQ ID No: 8-9):

The primer sequences are as follows:

BT FP: GAATTCATGGACAACAACCCGAACATCA

BT RP: ACTAGTTCAGTACTCGGCCTCGAAGGTC

The mCry1Ab gene was amplified by the pair of primers, and the amplified fragment and the vector pCAMBIA1391-SAFES6 and pCAMBIA1391-ACTIN were digested with EcoRI and SpeI, and the mCry1Ab fragment, pCAMBIA1391-SAFES6 vector fragment and pCAMBIA1391-ACTIN were separately recovered. A large fragment of the vector was ligated with the mCry1Ab fragment and the pCAMBIA1391-SAFES6 vector large fragment, and the mCry1Ab fragment and the pCAMBIA1391-ACTIN vector large fragment, respectively, using T4 DNA ligase to obtain the substitution of the Gus gene with mCry1Ab, by SAFES6. The expression vector driving Bt was named SAFES6-BT (Fig. 6); the expression vector for driving Bt by ACTIN was named ACTIN-BT (Fig. 7).

2. Quantitative analysis of BT

The SAFES6-BT vector and ACTIN-BT vector were transferred into Agrobacterium tumefaciens EHA105 by freeze-thaw method. The Agrobacterium-mediated rice genetic transformation method was used to obtain the corresponding vector transgenic plants, which was called SAFES6- BT plants and ACTIN-BT plants. For the specific method, refer to "Step 1 in Embodiment 3". The leaves, roots and endosperm samples of SAFES6-BT plants and ACTIN-BT plants were extracted and reverse transcribed into cDNA. The relative expression levels of BT genes in cDNA from different parts of the two plants were detected by qRT-PCR reaction. The quantitative qRT-PCR primers used to calibrate the internal reference gene ACTIN were as previously described (SEQ ID No: 4-5). The qRT-PCR primers used to detect BT gene expression were (SEQ ID No: 10-11):

BT upstream primer: 5'-ATCCCGCCGCAGAACA-3'

BT downstream primer: 5'-GAGCGGAACATGGACACG-3'

The results of the quantitative analysis are shown in Table 1. In the SAFES6-BT plants and ACTIN-BT plants, the Bt expression levels of the SAFES6-BT plants in the leaves and root tissues were slightly stronger or similar than those of the ACTIN promoter-driven Bt lines. In the endosperm, ACTIN-BT is expressed, while SAFES6-BT is not detected (below the detection line). It is indicated that the endosperm non-expression promoter SAFES6 of the present invention does not drive Bt expression or the expression level in the endosperm is very low and can be ignored.

Table 1

N.D., non-detected (below the detection line)

While the principles of the present invention have been described in detail, the preferred embodiments of the present invention The details in the examples are not intended to limit the scope of the invention, without departing from the invention. In the case of the spirit and scope, any obvious changes, such as equivalent transformations, simple substitutions, etc., based on the technical solutions of the present invention are within the scope of the present invention.

Claims (10)

An isolated endosperm does not express the promoter SAFES6, characterized in that the endosperm non-expressing promoter SAFES6 comprises at least one of the following polynucleotide sequences: (a) a polynucleotide sequence comprising the nucleotide sequence shown as SEQ ID NO: 1 in the Sequence Listing; (b) a nucleotide sequence having at least 85% homology to the nucleotide sequence shown by SEQ ID NO: 1 in the Sequence Listing and having the same function; (c) a nucleoside obtained by adding, substituting or deleting one or more nucleotides in the nucleotide sequence shown by SEQ ID NO: 1 in the Sequence Listing, and SEQ ID NO: 1 in the Sequence Listing a nucleotide sequence having the same function as the acid sequence; (d) a nucleotide sequence complementary to a nucleotide sequence comprising SEQ ID NO: 1 in the Sequence Listing, Wherein the endosperm non-expressing promoter SAFES6 is capable of ligating to a heterologous transcribable polynucleotide sequence and is capable of directing transcription of the heterologous transcribable polynucleotide sequence in non-endosperm tissue. An expression cassette comprising the promoter of claim 1 and a heterologous transcribable polynucleotide sequence linked to the promoter, wherein the promoter is capable of eliciting the Transcription and expression of a heterologous transcribable polynucleotide sequence in plant non-endosperm tissue transformed with the expression cassette. The expression cassette according to claim 2, wherein said heterologous transcribable polynucleotide sequence is a gene having crop expression characteristics, and said heterologous transcribable polynucleotide sequence is inserted into said expression cassette in a sense orientation, Alternatively, the heterologous transcribable polynucleotide sequence is inserted into the expression cassette in an antisense orientation. A recombinant expression vector comprising the promoter of claim 1 or the expression cassette of claim 2. The recombinant expression vector of claim 4, wherein the heterologous transcribable polynucleotide sequence is a gene having a function of altering a crop trait. A method for improving plant growth characteristics without altering plant endosperm traits, the method comprising: (1) obtaining the endosperm non-expression promoter SAFES6 according to claim 1; (2) linking the endosperm non-expression promoter SAFES6 to a gene having a function of altering crop traits to obtain a corresponding recombinant vector; (3) transferring the recombinant vector into a cell of a target plant; (4) cultivating the corresponding transgenic plants using the cells; (5) selecting, from the obtained transgenic plants, the gene having the function of changing crop traits expressed in the non-endosperm site, and the plant not expressing at the endosperm, the trait of the plant at the non-endosperm site is improved, and the endosperm site is not Affected. A method of transforming a plant or plant part, the method comprising transforming the plant or plant part with the expression cassette of claim 2 or the recombinant expression vector of claim 4. The method according to claim 7, wherein the plant part is selected from the group consisting of a cell, a protoplast, a cell tissue culture, a callus, a cell mass, a germ, a pollen, an ovule, Petals, styles, stamens, leaves, roots, root tips and anthers; said plants are selected from the group consisting of: food crops, vegetable crops, flower crops, energy crops. A method of directing expression of a transcribable polynucleotide sequence in a plant cell, the method comprising ligating the promoter of claim 1 to a heterologous transcribable polynucleotide sequence to construct a recombinant expression vector, The recombinant expression vector is transformed into plant cells. The method according to claim 9, wherein said transcribable polynucleotide sequence is used for transcription of the following proteins: anti-insect protein, antibacterial protein, antifungal protein, antiviral protein, anti-nematode protein, herbicide resistant Protein, an anti-stress protein, a selectable marker protein, the method further comprising regenerating the plant from the transformed plant cell.

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