BRPI1009936A2 - method of promoting the exacerbated increase in plant biomass - Google Patents

Abstract

METHOD OF PROMOTING THE EXACERATED RISE OF VEGETABLE BIOMASS. The invention relates to a method of promoting an exacerbated increase in plant biomass by 30 to 80% greater than unprocessed plants by introducing a polynucleotide sequence into the genome of a plant of commercial interest. The identifying sequence 1 (Seq.ld1) or the identifying sequence 2 (Seq.ld 2) encodes a gene belonging to the anaphase / cyclosome promoter complex (APC / C), and may be as well as homologous, orthologous or paralogous sequences.

Description

METHOD FOR PROMOTION OF EXACERATED VEGETABLE BIOMASS INCREASE

BACKGROUND OF THE INVENTION

Increasing agricultural productivity for both the food industry and the biofuel industry could solve the world's food shortage, thus supporting our growing population. Solve the problems we face and will still face for a long time, because our main source of energy (fuel), oil, is not renewable, as well as the decrease in pollution in the world, as we are talking about the increase in the production of biofuels.

In the last decades, the number of researches that aim to obtain genetically modified plants that present a more vigorous growth than that normally verified in the nature has been increasing.

The Belgian company CROPDESIGN is a leader in this line of research and holds several patent applications on this subject, including patent applications PI0513352-1, PI0514478, PI0317754, W005059147, W005117568 and W006005751.

PI0513352-1 describes a method for enhancing plant growth characteristics by increasing expression and / or activity in a plant, an LRR receptor kinase or a homolog thereof. This method comprises introducing into a mono or dicotyledonous plant an RLK827 nucleic acid molecule or functional variant thereof.

PI0514478-7 relates to a method of promoting plant growth by introducing into the plant the coding gene for an RNA ligand protein comprising 2 or 3 RNA recognition motifs containing the KIFVGGL and RPRGFGF polypeptide sequences.

PI0317754-8 relates to a method of promoting plant growth by modifying the expression of zinc finger 2xC2H2 protein in plants of commercial interest.

Referring to the PCT patent applications cited, we see that document W005059147 describes a method of promoting plant growth by increasing expression of the GRUBX protein coding gene. W005117568 describes plants with increased growth and method of obtaining them by inserting the gene encoding the protein E2F dimeriztion partner (DP) into the plant genome. The W006005751 application refers to a method of promoting plant growth by increasing the activity of the YIPPEE-like polypeptide or its homologous in plants of interest.

US6897359 (Senesco, Inc) protects a method of promoting tomato growth by inserting the deoxyhypine synthase gene into the tomato genome, which is also capable of making the tomato more pest resistant.

W007115064 (PIONEER HI-BRED INT.) Describes a method of modulating the size of a plant by expression in said plant of the ARGOS gene or homologues thereof.

All of these patent documents are capable of promoting a small or moderate increase in plant size, and some of these documents are effective only in specific plant organs and are not effective in promoting plant growth as a whole.

Anaphase-forming complex (APC or cyclosome) is the main ubiquitin linker necessary for the onset of anaphase, acting at the checkpoint between the end of metaphase and the beginning of anaphase. The cyclosome promotes the ubiquitination of proteins such as cyclins and securins, promoting their degradation by the 26S proteasome, both in mitosis and meiosis.

This complex is regulated by its phosphorylation, as well as by several other activators and inhibitors that alter its substrate specificity at different stages of the cell cycle. This "fine control" ensures timely degradation of cell cycle regulators.

Among the various components of the cyclosome, we have the proteins APC1, APC2, APC3, APC4, APC5, APC6, APC7, APC8 and others. Among these proteins, we see that APC3, APC6, APC7 and APC8 have several tetratricopeptide repeats (TPR) which are motifs of protein-protein interaction. Kraft et al (2003) demonstrated in vitro that APC3 and APC7 interact with the Cdc20 and Cdh1 IR motifs. In addition, APC3 and APC7 have 4 TRP subunits that account for most of the phosphorylation sites present in the APC / cyclosome complex.

APC7 is an Anaphase Promoter Complex gene, it has been described in vertebrates and plants, but has not been identified in yeast.

This gene was first incorrectly noted when the A. thaliana genome was sequenced (The Arabidopsis Genome Initiative1 2000) and only the C-terminal region with the NR-Iike protein was identified. It was later sequenced and correctly annotated as filed in the TAIR (www.arabidopsis.org) by access number At2g39090.

Tobacco has a gene, which encodes a 23 kDa protein with anti-viral activity called an inhibitor of virus replication (WfR) -like. This gene has high homology to the C-terminal region of one of the components of the anaphase-forming complex (APC or cyclosome), APC7. The cyclosome is a complex of proteins activated during mitosis, triggering the onset of anaphase. Cyclosome is an E3 ubiquitin ligase type protein that has the function of labeling proteins targeting degradation by 26S proteasome.

(NR) -like was first found in the culture medium of Nicotiana tabacum cv. Samsun NN (IVR protoplasts) infected with Tabacco mosaic virus (TMV) and this protein powerfully reduces TMV replication in assays with N. tabacum (AKAD et al, 2005).

Recently, in a paper presented by Park et al (2005), it has been shown that some types of breast cancer are involved with loss of expression of APC7, which is more common in cases of breast carcinoma with worse prognostic parameters or malignant features. They then suggest that dysregulation of APC activity, possibly due to low APC7 expression, may be associated with tumorigenesis in breast cancer.

The invention which will be described herein is capable of promoting an exaggerated growth of the vegetable as a whole, being visible its effects on organs such as leaf, stem and root. DESCRIPTION OF THE FIGURES

Figure 1: Scheme of multiplication of transformed plants. The transformed plants (T0) generated seeds by self-fertilization. The segregation of these seeds was determined (3: 1) and the plants passed to the earth (generation T1). The T1 generation plants generated seeds by self-fertilization (T2 generation). Homozygous and hemizygote plants of each strain were analyzed.

Figure 2: Comparison of exacerbated growth of transformed plants and control plants. Weight = average of 5 plants of each type.

Figure 3: Comparison of the number of fruits in the terminal portion of the branches found in transformed and control plants.

Figure 4: Comparison of leaf width found in transformed and control plants.

Figure 5: Comparison of leaf area found in transformed plants and control plants.

SUMMARY OF THE INVENTION

One of the objects of this invention is a transformation method of promoting the exacerbated growth of a plant by introducing and overexpressing a plant polynucleotide sequence (Seq. Id 1 and / or Seq. Id 2) into a plant's genome. belonging to the anaphase / cyclosome promoter complex (APC / C).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to the method of promoting exacerbated increase in plant biomass comprising introducing a polynucleotide sequence into the genome of a plant of commercial interest. Said polynucleotide sequence to be inserted into the genome of a plant of commercial interest must be linked to an overexpression vector and must be a polynucleotide sequence encoding a gene belonging to the anaphase promoter complex and / or a chimeric sequence containing a gene belonging to the anaphase promoter complex and a T-DNA sequence promoter of a directed insertional mutation, preferably the identifier sequence 1 (Seq.ldl) and / or the identifier sequence 2 (Seq.Id 2) may be inserted as well as their homologous, ortholog or paralog sequences. For this invention we have that homologous sequences are those sequences that encode proteins, enzymes, peptides or oligopeptides containing amino acid sequence substitutions, deletions and / or insertions, but which maintain their biological and / or functional activity similar to that of the unchanged protein. Paralogous sequences are sequences of the same species that derive from the duplication of an ancestral polynucleotide sequence; and, ortholog sequences are polynucleotide sequences found in different species, having originated in a speciation process from an ancestral polynucleotide sequence.

Preferably, said homologous, paralogous and orthologous sequences have a protein-level identity of between 50 and 95% when compared, aligned or not, to standard genomic comparison tools, for example, but not limited to BLASTp, Clustal and COBALT More preferably, the protein-level identity of homologous, paralogous and orthologous sequences should be between 75 and 90% when compared to identifier sequences 1 and / or 2.

Vegetables of commercial interest are vegetables of the monocotyledonous, dicotyledonous and eudicotyledonous families traditionally employed by humans in large-scale agriculture, such as vegetables in the group consisting of: maize, sugar cane, sorghum, soybeans, beans, tomatoes, tobacco, potatoes, sweet potatoes, rice, eucalyptus, cassava, oats and rye, but not limited to these species.

The method of this invention is obtained by constructing an expression vector, preferably an overexpression plasmid, where a polynucleotide sequence of interest must be inserted, which may be employed as Seq.Id 1 or Seq.Id 2, which will be overexpressed by after its insertion in the genome.

For this invention, we have that overexpression vector is one that contains a strong promoter sequence and selective genes. The promoter sequence of this invention may be the sequence of a specific or inducible constitutive promoter. Preferably, the overexpression vector is a suitable plasmid for plant transformation and the promoter sequence employed is a strong constitutive promoter sequence, which may be any of the group consisting of: nos promoter, 35S promoter, MPNPR1 promoter, ubiquitin I promoter, and / or the 19S promoter; and selective genes are genes that confer resistance to antibiotics and / or genes linked to β-galactosidase metabolism. Even more preferably, the vectors employed in this process are plasmids pk7WG2 (pk7WGF2 and / or pk7WG43DNew.

Several molecular biology techniques known to those skilled in the art may be employed to obtain the desired nucleotide sequences into the overexpression vector, for example, the techniques enable the following steps of the vector cloning process to be performed: RNA isolation total of a superior vegetable; selection of poly (A) + RNA fraction; cDNA synthesis (RT-PCR, real-time RT-PCR, quantitative real-time RT-PCR); cloning of transient vector sequence; specific stable vector sequence cloning. Preferably the higher plant employed in total RNA isolation belongs to Arabidopsis thaliana species.

For cDNA synthesis to be possible, the identification sequences 3 to 7 should be used as amplification initiators. According to the sequence to be amplified and later inserted into the overexpression vector, two of the listed primers are used. The examples will provide more clarity on this step of this patent object.

After insertion of the desired polynucleotide sequence into the expression vector, the following steps should be performed in order: transformation of a transient (maintenance) host to facilitate selection and multiplication of clones; definitive host transformation, plant transformation and regeneration of the transformed plant.

Any direct or indirect plant transformation techniques pertaining to the state of the art may be employed, such as transformation techniques belonging to the group consisting of: Agrobacterium tumefaciens conjugation, biobalistics, protoplast electroporation, floral dip and polycation protoplast transformation .

The sequences employed in this process of promoting plant overgrowth are sequences belonging to the APC / C complex and / or chimeric sequences containing a directed mutational sequence inserted into a sequence belonging to the APC / C complex. The identifying sequence 1 (Seq.Id. 1) refers to the complete sequence of a gene belonging to the anaphase forming complex (APC genes). The gene encoded by Seq.Id. 1 undergoes an alternative splicing that generates the identifier sequence 2 (Seq.Id. 2) which contains a high homology to the originally isolated tobacco-like gene (IVR). APC genes are described as having the function of regulating the process of cell division. It has also been shown that decreased or lost APC7 expression is involved in breast carcinoma. There is, however, no prior description describing that overexpression of APC genes in plants triggers the exacerbated plant growth process as employed by this patent application.

The plants transformed according to this process have an exaggerated growth, when compared to the control plants (unprocessed plants), growth related to the leaf size and plant size and mainly to the plant biomass. Notably, the biomass growth presented by the transformed plants is between 30 and 80% higher than the unprocessed ones. Preferably, the growth of plants transformed by the process described is 40 to 65% higher than that of unprocessed plants.

Therefore, this object of the invention should be employed in promoting the overgrowth of commercially important vegetables belonging to the monocotyledonous, dicotyledonous and eudicotyledonous families belonging to the group consisting of: corn, sugarcane, sorghum, soybeans, beans, tomatoes, tobacco, sweet potatoes, sweet potatoes, rice, eucalyptus, cassava, oats and rye, but not limited to these species.

This invention further relates to an overexpression vector suitable for transformation of a plant belonging to the monocotyledon, dicotyledon and eudicotyledon families, containing any of the identifying sequences 1 and / or 2 in addition to a strong promoter sequence and selective genes. The promoter sequence of this invention may be the sequence of a specific or inducible constitutive promoter.

Preferably, the promoter sequence employed is a strong constitutive promoter sequence, any one of which may be from the group consisting of: nos promoter, 35S promoter, MPNPR1 promoter, ubiquitin I promoter, and / or the 19S promoter. Preferably, airida, selective genes are those that confer resistance to antibiotics and / or genes linked to β-galactosidase metabolism. Most preferably, the vectors employed in this process are plasmids pk7WG2,0 and pk7WG43DNew.

The following examples are for illustrative purposes only and are intended to prove the embodiments of the inventors. These examples should not be used in delimiting the depositor's rights.

Example 1: Plant culture in vitro.

Example 1.1: Cultivation of Arabidopsis thaliana.

Arabidopsis thaliana ecotype Columbia plants were grown in pots containing autoclaved soil and vermiculite (2: 1) or in GM medium containing MS salts (Murashige and Skoog, 1962). For sterilization, Arabidopsis seeds, kept at 4 ° C, were placed in microcentrifuge tubes containing 1 mL of 70% ethanol for two minutes. After ethanol removal, the seeds were placed in a 5% sodium hypochlorite and 0.05% Tween 20 solution for 10 minutes. They were washed five times in autoclaved distilled water. The sterilized seeds were placed in solid culture medium with the aid of an autoclaved toothpick. Dormancy was broken at 4 ° C for at least three days. The plants were grown in a greenhouse at 21 ° C with photoperiod of 16 hours of light and 8 hours of dark.

Example 1.2: Cultivation of Nicotiana tabacum.

Nicotiana tabacum variety SR1 plants were grown in pots containing autoclaved soil and vermiculite (2: 1) or in plates and pots containing MS medium. For sterilization, the tobacco seeds, kept at 4 ° C, were placed in microcentrifuge tubes containing 1mL of 70% ethanol for two minutes. After ethanol removal, the seeds were placed in a solution of 5% sodium hypochlorite and 0.01% Tween for 15 minutes. They were washed five times in autoclaved distilled water. The sterilized seeds were placed in solid culture medium with the aid of autoclaved wooden sticks. The plants were grown in a greenhouse at 28 ° C with 12 hours light photoperiod under light intensity between 3,500-6,000 lux.

Example 2: Total RNA extraction and DNAse treatment.

Plants and organs were collected after the period of growth in the earth. The collected material was frozen in liquid nitrogen and kept at - 70 ° C. The material was macerated in liquid nitrogen and transferred to 1.5mL microcentrifuge tubes containing 500μL extraction buffer (200mM Tris-Cl pH 7.5; 100mM LiCI; 5mM EDTA; SDS 1%), 250μL phenol and 250μL of chloroform. The mixture was stirred for 1 minute and centrifuged at 20,000xg for minutes at 40 ° C. The aqueous phase was transferred to a new tube in which 1 volume of chloroform / isoamyl alcohol (24: 1) was added. The new mixture was stirred for 1 minute and centrifuged at 20,000xg for 15 minutes at 40 ° C. The aqueous phase was transferred to a new tube to which 1 volume of 6M lithium chloride was added. The tube was left at 4 ° C for approximately 16 hours. Subsequently the material was centrifuged at 20,000xg for 20 minutes at 40 ° C, the supernatant was discarded and the precipitate was resuspended in 1mL of 3M lithium chloride. The mixture was incubated at -70 ° C for 30 minutes and then centrifuged at 20,000xg for 15 minutes at 40 ° C. The supernatant was discarded and the precipitate washed with a 70% DEPC ethanol solution. Centrifuged at 20,000xg for 5 minutes at 40 ° C. Ethanol was discarded and the precipitated RNA was resuspended in 50μL of Milli-Q DEPC water.

Prior to cDNA synthesis, total RNA was treated with DNAse I (Promega) to eliminate any contamination with genomic DNA. The RNA was incubated with 0.5U DNAse per μg RNA1 in enzyme buffer (200mM Tris-Cl pH 8.3; 500mM KCI; 25mM MgCl2) at 37 ° C for 10 minutes, then pelleted. 300μL. RNA was purified by adding 1 volume of phenol and centrifuged at 20,000xg for 10 minutes at 40 ° C. The aqueous phase was transferred to a new tube to which 1 volume of chloroform was added. It was centrifuged at 20,000 xg for 10 minutes at 40 ° C and the aqueous phase was transferred to a new tube. RNA was precipitated by the addition of 1/10 volume of 3M sodium acetate and 2 volumes of absolute ethanol, followed by incubation at -70 ° C for 20 minutes. It was then centrifuged at 20,000xg for 20 minutes. The supernatant was discarded and the precipitated RNA was washed with a 70% ethanol DEPC solution. It is centrifuged for 5 minutes at 20,000xg and the supernatant is discarded and the precipitate is resuspended in 50 µl Milli-Q water. All solutions contained in the RNA extraction protocol were treated with 0.1% DEPC for 16 hours at room temperature.

After this period, the solutions were autoclaved.

Example 3: DNA Synthesis and Cloning

Example 3.1 Synthesis of the first cDNA tape.

Total RNA was treated with DNAse I and then a reverse transcription reaction was performed to obtain the first cDNA strand from mRNA. First strand syntheses were performed using 2 kits: "First-Strand cDNA Synthesis Pharmacia Kit" (GE healthcare) and "Taqman First strand cDNA synthesis kit" (Applied Biosystems).

Example 3.2: Cloning on Gateway Vectors.

The first cDNA strand synthesized using First-Strand cDNA Synthesis Pharmacia Kif was made in reactions with a final volume of 15μL according to the manufacturer. 2μg of total RNA was denatured at 65 ° C for 10 minutes in a volume of 8μL. After this denaturation period were added to the RNA: 1m L (0.2μg) of Not-dT oligonucleotides, 1μL of 200mM DTT and 5μL of Bulk Mix (cloned, FPLCpure Murine Reverse Transcriptase, RNAguard, RNAse / DNAse-Free BSA1). dATP, dCTP, dGTP and dTTP) The mixture was incubated for 1 hour at 37 ° C. Synthesized cDNAs were treated with 1U RNase H per reaction.

Oligonucleotide used:

Not-dT: 5'-AACTGGAAGAATTCGCGGCCGCAGGAAT18-3 '

Example 3.2.1: First Ribbon for Real-Time RT-PCR Analysis

The first cDNA strand synthesized using the 'Taqman First strand cDNA synthesis kit' was made in reactions with a final volume of 25μL, according to the manufacturer. For each 500ng of total RNA, 2.5μL of TaqMan RT Buffer 10X was added. 5.5μL 25mM MgCl2, 5μL dNTPs Mix, 1.25μL Random Hexamero, 0.5μL RNase Inhibitor, 0.625μL MultiScribe ™ Reverse Transcriptase (501) / μL The samples were incubated at 25 ° C for 10 minutes, followed by 48 ° C for 30 minutes and a final step of 95 ° C for 5 minutes The samples were diluted four times with 10mM Tris-Cl pH 8.0 and stored at -20 ° C or readily used.

Oligonucleotide Used: Random Hexamer (Applied Biosystems)

Example 3.3: Amplification of cDNA Molecule.

Real-time PCR was performed using the SYBR Green PCR Master Mix kit from Applied Biosystems, according to the manufacturer's recommendations.

The reaction was performed in a 96-well plate (MicroAmp Optical 96-Well Reaction Plate1 from Applied Biosystems). In each well were placed 6.25μL of the 4-fold diluted first reaction, 12.5μL of the kit's mix solution and 6.25μL of a mixture of the two oligonucleotides at 3.6μΜ each. For each reaction using the specific oligonucleotides for each gene, another was made with oligonucleotides specific for the Ubiquitin 14 gene (UBI14), a gene with approximately constitutive expression in all cells of the plant. This reaction was used as a positive control and as a normalizer for the amount of cDNA first strand used in the experiments. As a negative control, a reaction was performed under the same conditions as the positive control without the first ribbon mold. The 96-well plate is placed in a rack inside the PCR machine (ABI Prism 7700, Applied Biosystems). This bracket is responsible for changing the temperature on the board according to the established program. A tungsten-halogen lamp illuminates the plate. As SyBr in the mix is incorporated into the new DNA strands, it emits fluorescence when excited by lamp illumination. The light coming from the plate reflects through 4 optical filters, each with a different color (yellow, blue, green and red). A camera attached to the device converts the fluorescence intensity of colors into quantitative data. The ABl Prism 7000 SDS Software program brings together all the data components of each sample, determining the total intensity of each one. The raw result of the real-time PCR software is a logarithmic curve of the fluorescence intensity in the early phase, which gradually becomes a saturation plateau. In order to determine the precise quantity of the PCR product, it is essential that the measurement point is well determined. You need to disregard the initial signal, which may contain basal fluorescence. This is very evident in the early cycles, before there is a significant amount of amplified DNA. It is based on these cycles that the basic fluorescence line of the samples is determined. The measurement point chosen was above the baseline, still in the logarithmic phase of the curve.

The measuring point determines the number of cycles required to generate fluorescence at the chosen point. The values obtained are transformed to a linear scale, using base 2 as two DNA strands (exponential reaction). The result obtained is the amount of fluorescence found at that given number of cycles in arbitrary unit. The fluorescence value of each sample with the specific oligonucleotides of the genes in question is then divided by the fluorescence of the corresponding sample with the UBI14 oligonucleotide. Thus, the samples are normalized by the amount of first tape. After this calculation is made, the value of each sample is divided by the value of the control sample. Thus we get the value of how many times the sample expresses the gene in question in relation to the control of the experiment.

Fl. Rel. = 2 (CT Ubi-CT gene)

Where, Fl. Rel. Is the relative fluorescence. CT ubi is the average number of cycles at the chosen control point with UBI14.

CT gene is the average number of cycles at the chosen point of the curve in question.

For relative expression of experiments with experimental controls, then: Fl. Exp / Fl. Count Count

EXAMPLE 3.4: Cloning Using the Gateway® System (Invitrogen®)

While any type of commercially available cloning systems may be used, we will only exemplify cloning performed using the Gateway® (Invitorgen®) system. This system is based on the transfer of one or more DNA sequences to multiple vectors in parallel reactions while maintaining the same orientation and reading code. For this, the technique uses specific recombination sites (att) of phage λ and a set of enzymes called recombinases. The cloning system consists of two steps: the LR reaction and the BP reaction. The BP reaction is a recombination reaction, catalyzed by the BP Clonase Enzyme Mix, between a PCR product or an expression clone and the donor vector (pDONR201 and pDONR221). This reaction serves to create an entry-clone from a donor vector. From the input clone one can transfer the DNA to any Gateway vector. donor vector, recombination regions need to be added to specific oligonucleotides, so the end product of the PCR reaction will be flanked by the attB1 and atfB2 recombination sequences. When the BP reaction occurs, the donor vector (which has affP1 and attP2 regions) recombine with the PCR product (which has attB1 and attQ2 regions) and will form an input vector containing the A 1L1 and attL2 regions (Figure XX) This ensures the correct orientation of the ORF after the reaction.

The conditions used in amplifying AtAPC7 and AtAPC7 splicing cDNAs were (Table 1) (1st PCR):

<table> table see original document page 14 </column> </row> <table>

Table 1: 1st PCR reaction protocol for cloning in Gateway vectors.

Pfx enzyme buffer (50mM Tris-Cl pH 8.0, 50mM KCI, 1mM DTT, 0.1mM EDTA, stabilizers and 50% glycerol).

The reaction occurred in a Mastercycler® (Eppendorf) thermal cycler using the following conditions (table 2): <table> table see original document page 15 </column> </row> <table>

Table 2: Parameters used in the 1st PCR reaction.

PCR amplifications of some ORFs of this work were performed using only one pair of specific oligonucleotides containing the entire attB recombination region adapter sequence. With the inclusion of the recombination region in the specific oligonucleotides, only one PCR reaction is required (60 nucleotide oligonucleotides).

However, for the amplification of some ORFs, a second PCR reaction was performed using the first PCR product as a template. Oligonucleotides containing the adapter region, including part of the region in the first pair of specific oligonucleotides, were used to generate an end product containing the recombination region (attB).

In the second amplification (table 3) (2nd PCR), the following parameters were used:

<table> table see original document page 15 </column> </row> <table>

Table 3: Parameters used in the 2nd PCR reaction.

The DNA fragment of interest, after purification, was cloned into the pDONR201 / 221 vector (Invitrogen) in a reaction called BP (Table 4). The BP reaction was incubated at 25 ° C for 12 hours and then dialyzed for 3 hours in 0.025μm membrane (Millipore). After dialysis, the reaction was used to transform E. coli DH5a by electroporation. Colonies resistant to 50μg / mL kanamycin were inoculated into liquid LB medium plus this antibiotic. Plasmid DNA extraction was performed using the "GFX Piasmid Minipreparation" kit (GE Healthcare) following the manufacturer's instructions. Once confirmed by digestion and sequencing, this new plasmid is called an input clone.

<table> table see original document page 16 </column> </row> <table>

Table 4: Components of the BP reaction. Reactions with final volume of 5μL.

The LR reaction occurred from the input clone and the DNA can be transferred to any Gateway expression vector. The LR reaction is precisely the recombination between the system input vector and the target vector (Figure 10). This reaction is catalyzed by the "LR Clonase Enzyme Mix". The attL1 and atL2 regions present in the input vector (formed by the BP reaction) and the attR1 and attR2 regions present in the target vectors will be recombined and the expression clone will appear. The resulting expression clone has the affB1 and attB2 sites identical to those present in PCR products. In this work, the target vectors used were pK7WG2, pK7WGF2 and pK7GW43DNew.

The LR reaction should be incubated at 25 ° C for 12 hours and then dialyzed for 3 hours in 0.025μm membrane (Millipore). After dialysis, the reaction was used to transform E. coli DH5a by electroporation. Antibiotic resistant colonies required by vector selection were inoculated into liquid LB medium plus specific antibiotics. Plasmid DNA extraction was performed using the "GFX Plasmid Minipreparation" kit (GE Healthcare) following the manufacturer's instructions. Once confirmed by digestion, this new plasmid is now called the target clone.

Donor vectors and target vectors contain the ccdB gene flanked by the recombination regions. By coding a lethal protein for the bacteria used in the experiments, it works as a false positive inhibitor. If recombination does not occur, bacteria transformed with these vectors will fail at the time of DNA replication. Thus, any recombination failure between the vectors will not result in viable clones.

Example 3.4.1: Proof of Amplification:

The PCR reaction products, digestions and ligations were electrophoretically separated on 1.0% agarose gel prepared in TAE buffer (80mM Tris-base; 80mM acetic acid; 2mM EDTA pH 8.0) and ethidium bromide (0, 5pg / mL). The gel was placed in the i-Mupid® electrophoresis vat, immersed in 0.5X TAE and subjected to 100V for 30 minutes.

Example 3.4.2: DNA Extraction from Agarose Gel:

To purify the DNA fragment of the gel, the GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) was used according to the manufacturer's instructions.

The desired bands were cut from the gel with the aid of a scalpel blade. These bands were placed in a 1.5mL microcentrifuge tube. For each 10mg of gel, 10μL of capture buffer was added and the tubes incubated at 60 ° C (until the agarose was completely dissolved). The sample was transferred to the GFX column coupled to a 1.5mL microcentrifuge tube and incubated for 1 minute at room temperature. The tube / column system was then centrifuged for 30 seconds at 20,000g and the liquid discarded. 500μL of wash buffer was added to the purification column and then centrifuged for 30 seconds at 20,000g. The column was transferred to a new 1.5mL microcentrifuge tube. 30μL of Milli-Q water was added to the column. After centrifugation, the solution containing the gel-eluted DNA was estimated by 1% agarose gel electrophoresis.

Example 3.4.3. Agrobacterium tumefaciens transformation:

The transformation of Agrobacterium tumefaciens is a sudden change in temperature (characterizing thermal shock). 1μg of DNA was added to the aliquot of competent cells (200μL). The microcentrifuge tube containing the binding and the cells were placed on ice for 30 minutes then placed in liquid nitrogen for 2 minutes. Then the tube was incubated at 37 ° C for 5 minutes. The solution was transferred to a 15mL tube containing 1mL YEB and incubated for 1 hour at 30 ° C with shaking. At the end of the period, the cell suspension was seeded in a plate containing solid YEB with the appropriate antibiotic.

Example 3.4.3 (b). Genetic transformation of Arabidopsis thaliana - Columbia Ecotype

To obtain transfected plants of Arabidopsis thaliana, the in vivo transformation method (Floral dip) (CLOUGH and BENT, 1998) was used, using A. tumefasciens (C58C1Rif - PMP90 strain). Briefly, A. thaliana seeds of the Columbia ecotype were incubated for 2-3 days at 4 ° C in 27.5 mL of 0.2% agar. They were then germinated for two weeks in a sand: soil (1: 3) mixture and the seedlings obtained were transplanted to individual pots. Each pot with a 7-10 cm inflorescence height was used for transformation. The inflorescences were inverted and shaken for 3 sec in a culture of Agrobacterium tumefaciens transformed with the clones of interest. The plants were then maintained at 22 ° C / day, 18 ° C / night with a 12h light and 12h dark photoperiod in an Aracon system. Five weeks after transformation, the plants were transferred to a temperature of 25 ° C and, after two weeks, the seeds were collected and stored at 4 ° C for further analysis of F2 generation. The transformed material was sown in specific selective medium.

The following analyzes were performed on these transformants, always purchased with the control: leaf shape, flower structure, silica and seed structure and root growth.

Example 3.5. Genetic transformation of Nicotiana tabacum variety SR1:

To study the role of Seq.ld. 1 and Seq.ld 2 in plant development, the overexpression vector pK7GW43DNew was chosen. The vector has the cauliflower mosaic virus 35S promoter, a HA tail, and regions called LB and RB. The 35S promoter contains many regulatory elements, which allows a high rate of gene transcription. The LB and RB regions flank the vector overexpression region (35S :: ORF :: HA) and are required for the transfer of this vector functional unit to the plant genome via Agrobacterium sp. Prior to tobacco transformation, a pre-inoculum containing Agrobacterium tumefaciens transformed with the appropriate antibiotics in YEB medium must be made. The pre-inoculum should be kept at 28 ° C under agitation for 48 hours. The bacteria used for tobacco processing contained the overexpression constructs of SEq.Id 1 and Seq.Id. 2 of Arabidopsis thaliana. The antibiotics used in the pre-inocula were Sp, Sm, Rf and Gm.

The plants used were from the variety SR1 of Nicotiana tabacum. In laminar flow, the tobacco leaves were cut with a sterile scalpel in the form of a 1cm2 square without central rib fragments. These leaf explants were exposed for 10 minutes to their inoculum. After this period, the explants were placed on the surface of solid MS plates containing the hormones IAA and 6BA. The plates were left in the culture room in vitro for 2 days at 28 ° C and 12 hours photoperiod. Subsequently, the explants were transferred to a new solid MS plate containing IAA, 6BA, cefotaxime and kanamycin (cefotaxime antibiotic acts by inhibiting the growth of Agrobacterium sp and kanamycin antibiotic acts by selecting the transformed plants). At the end of the transfer, the plates were again placed in the culture room in vitro until calluses appeared on the explants surface. The calli were separated and transferred to a new MS medium containing IAA (rooting induction and shoot growth from each callus). After the development period in MS / IAA medium, the plants were transferred (vegetative propagation) to MS / Km selective medium. Rooting plants were transferred to pots containing soil and vermiculite (2: 1). These vessels were placed in the room in vivo at 28 ° C under 12 hour photoperiod. It is noteworthy that untransformed SR1 leaf explants (control) also underwent tissue culture (MS / IAA / 6BA medium and MS / IAA medium) until transferred to pots containing soil and vermiculite (2: 1).

Example 4. Obtaining transformed plants:

After obtaining the in vitro transformed strains (TO generation), plants overexpressing Seq.ld 1 and Seq.ld 2 were transferred to soil until they generated seeds by self-fertilization (T1 generation). The seeds were collected, sterilized and stored at 4 ° C. From the sowing of 100 seeds of each strain transformed in MS / Km, it was possible to determine the Mendelian segregation. To verify whether or not the selector agent was influencing the phenotype, 50 seeds were sown in DM without antibiotic. From the MS / Km plate, 5 plants of each strain were transferred to the pot containing soil and vermiculite in the house in vivo. Self-fertilization of these plants resulted in seeds corresponding to T2 generation. These seeds were sterilized, stored at 4 ° C and sown in MS / Km medium to determine their segregation (homozygotes or hemizigotes), see Figure XX.

Example 5. Verification of fresh and dry mass of tobacco plants:

The leaves and stem of each tobacco were collected and immediately weighed on a precision scale. The obtained results represented the values of fresh mass. Subsequently, the plant material was incubated in a drying oven at 70 ° C for 3 days. The dried material was weighed and the values corresponded to the dry mass.

Example 6. Leaf area calculation:

The length and width of the leaf blades were measured with the aid of a ruler. Length was measured from leaf tip to petiole. The measured width was the largest distance, ie the central region of the leaf. Results were multiplied to obtain gross leaf area.

Example 7. Statistical analysis of tobacco plants:

Statistical analyzes were performed using GraphPad Prism version 3 software (GraphPad Software Incorporated). Each statistical analysis was performed with a number of samples equal to 12 (plants) per building / lineage. The extreme values obtained (maximum and minimum values) were discarded. The experiments were performed in duplicate. The obtained data were submitted to the analysis of variance and the Turkey test for comparison of means. The Turkey test allows to establish the minimum significant difference, that is, the smallest difference in sample means that should be taken as statistically significant at a given level.

Example 8. Agroinfiltration Experiment:

Inoculum of constructs containing Seq.Id 1 and Seq.Id 2 and TMV :: GFP in A. tumefaciens were made in YEB medium with the respective antibiotics and incubated at 28 ° C for 48 hours. The culture was centrifuged at 4 ° C for 3 minutes at 3,000g rpm. The supernatant was discarded and the precipitate resuspended in 10mM MgSO4. The culture was again centrifuged at 4 ° C for 3 minutes at 3,000g rpm. The supernatant was discarded and the precipitate diluted in acetoceringone solution (1-2 μL / 10mL) until reaching D0600 = 1.0. The preparation was kept for 3 hours at room temperature before agroinfiltrating.

Plants of N.tabacum variety Samsun nn with 45 days were agroinfiltrated as follows:

Day 1: Inoculum of TMV :: GFP construction;

Day 3: Inoculum of constructs containing Seq.Id 1 and Seq.Id 2 (distinct treatments);

Day 5: Analysis of plants under ultraviolet light to see the level of viral infection;

Day 10: Analysis of plants under ultraviolet light to see level of viral infection;

Day 15: Analysis of plants under ultraviolet light to see level of viral infection;

All treatments were done in triplicate and as control of the experiment only A. non-transformed A. tumefaciens and A. non-transformed A. tumefaciens were agroinfiltrated followed by agroinfiltration of the constructs containing a Seq.Id 1 and Seq.Id 2 (separate treatments).

Example 9. DNA Sequence Analysis:

Nucleotide and amino acid sequence analyzes were performed using the Basic Local Alignment Search Toof (BLAST) suite of programs available on the Internet from the National Center for Bioinformatics and Information (NCBI) - http://www.ncbi.nlm.nih.gov ) (Altschul, et al, 1997) and the Clustal W multi-alignment tool (www .ebi.ac.uk / clustalw /).

The primary protein sequence was analyzed for homology through the BLASTp program (www.ncbi.nlm.nih.gov/BLAST) (Atschul, 1997). The Pfam program (www.pfam.wustl.edu) was used as a tool to find domains with established biological function.

For the construction of phylogenetic trees, the Molecular Evolutionary Genetics Analyzes program, version 3 - Mega 3 (Kumar et al, 2004) was used. Searches in the Arabidopsis genome were done using the tools available in A. thaliana's TAIR database (www.arabidopsis.org). sequences_ST25 SEQUENCE LISTING

<110> RIO DE JANEIRO FEDERAL UNIVERSITY

<120> METHOD FOR PROMOTING THE EXACREBED VEGETABLE BIOMASS INCREASE

<130> plant growth

<160> 2

<170> Patentln version 3.5

<210> 1

<211> 1757

<212> DNA

<213> Arabidopsis thaliana

<400> 1

atggaggttc caaaggagca gatcgcgact ttaatagagc atgggcttta cgattctgct 60 gaaatgctcg gctgctttct cgtttcttct cctactgtta gtgccgaaac tagtcctcag 120 ctcaaggcgg agaatttgat tctactgggt gatgctttat ttcatcagag agaacaccgg 180 agagctattc atacgtacaa gcaagcgttg catcattaca caaggattcc gaagcaaagc 240 tccggtattt cgaggagttc attatcttta tctaccagat catctgtgaa tgcctccagc 300 atttctgcta ttaatgagaa cgaggtgaga ttcaagattg cttcatctca ctttgctctt 360 aatgaaacaa aagctgcgat tgctgagatg gaatctgtca agaccaggag cttagagatg 420 aatatactga tggcaaagct tcatcgaaat tctggataca accgtggtgc tattgctttt 480 tataaagagt gtttaaggca gtgtccttat gtacttgaag ccgtcatagg tttagctgaa 540 ctgggagtta gtgcaaagga tatcatatca tcgtttactc agacttcaaa tagaagtgca 600 aaggtttcac tcgatcagat agatcctacc cgctggttgc aacgttatgt agaggcccag 660 tgttgtgttg cttcgcatgc ttacaaaggg gcgctggaac tctttgctga actcttacaa 720 cgatttccaa ataatgtaca cttgttgact gagacagcaa aggttgaagc cattattggg 780 aaaaatgatg aggccataat gagatttgag aaggttcggt caattgatcc ttacacacta 840 accagtatgg atgagtatgc gatgctgctt cagattaagt gtgattattc caggctaaac 900 aagctcgtcc acgatttatt aagcgttgat cataccagag cagaagtatt tgttgctttg 960 tctgtactat gggaaaggaa agatgcaagg acggcattat cttatgctga gaagagtatc 1020 agggtagacg agaggcacat acccggctac ataatgaagg gaaatctact tttacaagca 1080 aaacgaccag aagctgcagc gatcgccttc agggctgccc agaatttgag gtccgatctt 1140 cgttcatatc aaggcttagt ccattcttat cttgcatttg gtaaaaccaa agaagcattg 1200 tataccgcca gggaagcaat gaatgcaatg cctcaatccg cgaaggctct gaaattagtt 1260 ggtgatgttc atgcgtttac atcaagtggc agggaaaagg caaaaaagtt ttacgagtca 1320 ggtctgaggc tggaacctgg gtaccttgga gctgtgttag ccctagctga gcttcatcta 1380 atggaaggga ggaatggaga tgctgtatca ctacttgaac gatatctcaa agattatgca 1440 gatgattctc tccacgtcaa gctagctcaa gtgtttgctg caacgaatat gctacaagat 1500 tctttatcac actttcaagc tgcattaaga sequences ataaatccac ST25 agaatgaggc agccaaaaag 1560 ggactagatc gcttggagaa acaaatgaag ggaatagac cagatgcaac agatgagaat 1620 gacgagaacg atgttgaaga tgttgatgga gacaccgaag aagctgagct catgtgaaga 1680 gtagagaga c agaagagtgt gtaaactgta aaactatcga atccttaaca ttaatattaa 1740 tagcaaatgt cattcct 1757 <210> 2 <211> 911 <212> DNA <213> Arabidopsis thaliana <400> 2 atggatgagt atgcgatgct gcttcagatt aagtgtgatt attccaggct aaacaagctc 60 gtccacgatt tattaagcgt tgatcatacc agagcagaag tatttgttgc tttgtctgta 120 ctatgggaaa ggaaagatgc aaggacggca ttatcttatg ctgagaagag tatcagggta 180 gacgagaggc acatacccgg ctacataatg aagggaaatc tacttttaca agcaaaacga 240 ccagaagctg cagcgatcgc cttcagggct gcccagaatt tgaggtccga tcttcgttca 300 tatcaaggct tagtccattc ttatcttgca tttggtaaaa ccaaagaagc attgtatacc 360 gccagggaag caatgaatgc aatgcctcaa tccgcgaagg ctctgaaatt agttggtgat 420 gttcatgcgt ttacatcaag tggcagggaa aaggcaaaaa agttttacga gtcaggtctg 480 aggctggaac ctgggtacct tggagctgtg ttagccctag ctgagcttca tctaatggaa 540 gggaggaatg gagatgctgt atcactactt gaacgatatc tcaaagatta tgcagatgat 600 tctctccacg tcaagctagc tcaagtgttt gctgcaacga atatgctaca agattcttta 660 tcacactttc aagctgcatt aagaataaat ccacagaatg aggcagccaa aaagggacta 720 g atcgcttgg agaaacaaat gaagggaata gacccagatg caacagatga gaatgacgag 780 aacgatgttg aagatgttga tggagacacc gaagaagctg agctcatgtg 840 agacagaaga gtgtgtaaac tggaaccta 900t

SEQUENCE LISTING

<110> RIO DE JANEIRO FEDERAL UNIVERSITY

<120> METHOD FOR PROMOTING EXACERATED VEGETABLE BIOMASS INCREASE <13O> Plant Growth <160> 2

<170> PatentIn version 3.5

<210> 1 <211> 1757 <212> DNA

<213> Arabidopsis thaliana <400> 1

atggaggttc caaaggagca gatcgcgact ttaatagagc atgggcttta cgattctgct

60

gaaatgctcg gctgctttct cgtttcttct cctactgtta gtgccgaaac tagtcctcag 120

ctcaaggcgg agaatttgat tctactgggt gatgctttat ttcatcagag agaacaccgg 180

agagctattc atacgtacaa gcaagcgttg catcattaca caaggattcc gaagcaaagc 240

tccggtattt cgaggagttc attatcttta tctaccagat catctgtgaa tgcctccagc 300

atttctgcta ttaatgagaa cgaggtgaga ttcaagattg cttcatctca ctttgctctt 360

aatgaaacaa aagctgcgat tgctgagatg gaatctgtca agaccaggag cttagagatg 420

aatatactga tggcaaagct tcatcgaaat tctggataca accgtggtgc tattgctttt 480

tataaagagt gtttaaggca gtgtccttat gtacttgaag ccgtcatagg tttagctgaa 540

ctgggagtta gtgcaaagga tatcatatca tcgtttactc agacttcaaa tagaagtgca 600

aaggtttcac tcgatcagat agatcctacc cgctggttgc aacgttatgt agaggcccag 660

tgttgtgttg cttcgcatgc ttacaaaggg gcgctggaac tctttgctga actcttacaa 720 cgatttccaa ataatgtaca cttgttgact gagacagcaa aggttgaagc cattattggg aaaaatgatg aggccataat gagatttgag aaggttcggt caattgatcc ttacacacta accagtatgg atgagtatgc gatgctgctt cagattaagt gtgattattc caggctaaac

780

840

900 aagctcgtcc acgatttatt aagcgttgat cataccagag cagaagtatt tgttgctttg 960

tctgtactat gggaaaggaa agatgcaagg acggcattat cttatgctga gaagagtatc 1020

agggtagacg agaggcacat acccggctac ataatgaagg gaaatctact tttacaagca 1080

aaacgaccag aagctgcagc gatcgccttc agggctgccc agaatttgag gtccgatctt 1140

cgttcatatc aaggcttagt ccattcttat cttgcatttg gtaaaaccaa agaagcattg 1200

tataccgcca gggaagcaat gaatgcaatg cctcaatccg cgaaggctct gaaattagtt 1260

ggtgatgttc atgcgtttac atcaagtggc agggaaaagg caaaaaagtt ttacgagtca 1320

ggtctgaggc tggaacctgg gtaccttgga gctgtgttag ccctagctga gcttcatcta 1380

atggaaggga ggaatggaga tgctgtatca ctacttgaac gatatctcaa agattatgca 1440

gatgattctc tccacgtcaa gctagctcaa gtgtttgctg caacgaatat gctacaagat 1500

tctttatcac actttcaagc tgcattaaga ataaatccac agaatgaggc agccaaaaag 1560

ggactagatc gcttggagaa acaaatgaag ggaatagacc cagatgcaac agatgagaat 1620

gacgagaacg atgttgaaga tgttgatgga gacaccgaag aagctgagct catgtgaaga 1680

gtagagagac agaagagtgt gtaaactgta aaactatcga atccttaaca ttaatattaa 1740

tagcaaatgt cattcct 1757

<210> 2

<211> 911

<212> DNA

<213> Arabidopsis thaliana

<400> 2

atggatgagt atgcgatgct gcttcagatt aagtgtgatt attccaggct aaacaagctc

60

120

gtccacgatt tattaagcgt tgatcatacc agagcagaag tatttgttgc tttgtctgta

ctatgggaaa ggaaagatgc aaggacggca ttatcttatg ctgagaagag tatcagggta 180

gacgagaggc acatacccgg ctacataatg aagggaaatc tacttttaca agcaaaacga 240

ccagaagctg cagcgatcgc cttcagggct gcccagaatt tgaggtccga tcttcgttca 300 tatcaaggct tagtccattc ttatcttgca tttggtaaaa ccaaagaagc attgtatacc 360

gccagggaag caatgaatgc aatgcctcaa tccgcgaagg ctctgaaatt agttggtgat 420

gttcatgcgt ttacatcaag tggcagggaa aaggcaaaaa agttttacga gtcaggtctg 480

aggctggaac ctgggtacct tggagctgtg ttagccctag ctgagcttca tctaatggaa 540

gggaggaatg gagatgctgt atcactactt gaacgatatc tcaaagatta tgcagatgat 600

tctctccacg tcaagctagc tcaagtgttt gctgcaacga atatgctaca agattcttta 660

tcacactttc aagctgcatt aagaataaat ccacagaatg aggcagccaa aaagggacta 720

gatcgcttgg agaaacaaat gaagggaata gacccagatg caacagatga gaatgacgag 780

aacgatgttg aagatgttga tggagacacc gaagaagctg agctcatgtg aagagtagag 840

agacagaaga gtgtgtaaac tgtaaaacta tcgaatcctt aacattaata ttaatagcaa 900

atgtcattcc t 911

Claims (20)

1. A method for promoting the exacerbated increase in plant biomass, comprising the introduction of a polynucleotide sequence belonging to the directed anaphase promoter complex in the genome of a plant of commercial interest. 2. A method for promoting exacerbated increase in plant biomass, comprising introducing a chimeric polynucleotide sequence containing a gene belonging to the anaphase promoter complex and a T-DNA sequence promoting an insertional mutation directed into the genome of a plant. commercial interest. Method according to Claims 1 and 2, characterized in that the identifying sequence 1 (Seq.Idl) is inserted into the genome of a plant belonging to the monocotyledon, dicotyledon and eudicotyledon families traditionally employed by humans in large-scale agriculture. Method according to Claims 1 and 2, characterized in that the identifying sequence 2 (Seq.Id2) is inserted into the genome of a plant belonging to the monocotyledonous, dicotyledonous and eudicotyledon families traditionally employed by humans in large-scale agriculture. Method according to Claims 1 and 2, characterized in that it is used for the promotion of vegetables belonging to the group consisting of: corn, sugar cane, sorghum, soybean, beans, tomatoes, tobacco, potato, sweet potato , rice, eucalyptus, cassava, oats and rye. Method according to claims 1 and 2, characterized in that plasmids pk7WG2, pk7WGF2 and pk7WG43DNew are employed as vectors. Method according to claims 1 and 2, characterized in that the total RNA of Arabidopsis thaliana is isolated; cDNA synthesis; transient vector cloning; specific stable vector cloning. Method according to claim 7, characterized in that Seq.Id 3 is used for cDNA amplification. Method according to claim 7, characterized in that Seq.Id 4 is used for cDNA amplification. Method according to claim 7, characterized in that Seq.Id 5 is used for cDNA amplification. Method according to claim 7, characterized in that Seq.Id 6 is used for cDNA amplification. Method according to claim 7, characterized in that Seq.Id 7 is used for cDNA amplification. A method according to claims 1 and 2, characterized in that conjugation with plant transformation techniques belonging to the group consisting of: Agrobacterium tumefaciens, biobalistics, electroplating of protoplasts, floral dip and transformation of protoplasts with polycations is employed. Method according to Claims 1 and 2, characterized in that it is employed to promote the exacerbated growth of commercially important vegetables belonging to the monocotyledonous, dicotyledonous and eudicotyledonous families belonging to the group consisting of: corn, sugar cane, sorghum , soybeans, beans, tomatoes, tobacco, potatoes, sweet potatoes, rice, eucalyptus, cassava, oats and rye. 15. Overexpression vector suitable for the transformation of a plant belonging to the monocotyledonous, dicotyledonous and eudicotyledonous families, characterized by containing the identifying sequence 1 and a strong promoter sequence and selective genes. 16. Overexpression vector suitable for the transformation of a plant belonging to the monocotyledonous, dicotyledonous and eudicotyledonous families, characterized by having the identifying sequence 2 and a strong promoter sequence and selective genes. Vector according to claim 16, characterized in that it is plasmid pk7WG2,0 containing Seq.Id.:1. Vector according to claim 16, characterized in that it is plasmid pk7WG2,0 containing Seq.Id.:2. Vector according to claim 16, characterized in that it is plasmid pk7WG43DNew containing Seq.Id.:1. Vector according to claim 16, characterized in that it is plasmid pk7WG43DNew containing Seq.Id.:2.