BRPI1009965A2 - METHOD FOR PRODUCTION OF ENVIRONMENTAL STRESS-tolerant plants, their uses and recombinant DNA vector - Google Patents

Abstract

METHOD FOR PRODUCING TOLERANT PLANTS FOR ENVIRONMENTAL STRESS, ITS USES AND RECOMBINANT DNA VECTOR. Plants are influenced by a large number of biotic and abiotic environmental factors and recurrently abiotic stresses are more severe, affecting all plant functions, resulting in reduced growth and productivity. In this sense, the identification and understanding of abiotic tolerance mechanisms are fundamental in the development of new drought tolerant cultivars. Thus, the present invention provides a plant production method which contains in its cells a sequence of sugarcane nucleotides and overexpression of this gene leads the plant in question to greater tolerance to abiotic stresses. More broadly, a polynucleotide encoding the sugarcane protein which is expressed by a plant-working promoter and terminator is described. It is found that this gene confers tolerance to different abiotic stresses.

Description

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"METHOD FOR PRODUCING TOLERANT PLANTS FOR ENVIRONMENTAL STRESS, ITS USES AND RECOMBINANT DNA VECTOR"

FIELD OF INVENTION

Plants are influenced by a large number of environmental, biotic and abiotic factors and recurrently abiotic stresses such as drought, salinity, temperature, pollution, radiation and so on are more severe and affect all plant functions resulting in reduced growth and of productivity. It is estimated that this type of stress generally affects physiological, biochemical and morphological levels, and may reduce productivity by 50% and in some cases up to 70%. This has led to efforts in understanding the response of plants to stress.

In this sense, the identification and understanding of abiotic tolerance mechanisms are fundamental in the development of new cultivars tolerant to different abiotic stresses. Thus, the present invention describes a method for producing transgenic plants tolerant to environmental stresses by introducing a vector comprising a gene encoding new sugarcane protein of previously unknown function, in addition to constructing the vector. and its uses. -. »

BACKGROUND OF THE INVENTION

One of the main problems in agriculture is drought, which is by far the most important environmental stress. Many efforts have been made to improve plant productivity under water-limiting conditions. In recent decades, heavy investments have been made in the production of stress-tolerant transgenic plants, including drought and salt stress. This has allowed a better understanding of the physiological and molecular responses of plants to stress, opening perspectives to increase yield under stress conditions.

Among the technologies for tolerant plant production, the transformation by

Agrobacterium (Hooykaas PJ, Schilperoort RA (1992) Agrobacterium and plant genetic engineering. Plant Mol Biol 19: 15-38; Barton KA, Chilton MD (1983) Agrobacterium Ti plasmids as vectors for plant genetic engineering. Methods Enzymol 101: 527-539 ), direct gene transfer in protoplasts (Gharti-Chhetri GB, Cherdshewasart W, Dewulf J, Paszkowski J, Jacobs M, et al. (1990) Hybrid genes in the analysis of transformation conditions. 3. Temporal / spatial fate of NPTII gene integration, its inheritance and factors affecting these processes in Nicotiana plumbaginifolia Plant Mol Biol 14: 687-696; Lyznik LA, Peng JY, Hodges TK (1991) Simplified procedure for transient transformation of plant protoplasts using polyethylene glycol treatment. Rodenburg KW, Groot MJ, Schilperoort RA, Hooykaas PJ (1989) Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts Plant Mol Biol 13: 711-719 and Bailas N, Zakai N, Loyter A (1 987) Transient expression of pCaMVCAT plasmid in plant protoplasts following transformation with polyethyleneglycol. Exp Cell Res 170: 228-234) and the

Particle Bombardment (Klein TM, Wolf ED, Sanford JC (1987) High-velocity

------ - microprojectiles for-delivering.nucleic acids.into living.cells. Nature, 327: 70-73) are the most

used. .... .

In recent years great advances have been made in technologies for the transformation of

Plantation and research have focused on the need to develop effective methods of genetic transformation of the main species of experimental and economic interest. Agrotransformation has gained wide acceptance for the ease of the transformation method and the high rate of transgenic plants generated from this method. On the other hand, tobacco has been widely used as a gene transformation tool due to its ease of transformation and rapid generation of transformed generations. Thus, genes of different species can be initially evaluated in tobacco, for the ease and speed in the production and analysis of transgenic plants. This has occurred with genes from cereals, vegetables, ornamentals, medicinal plants, fruit trees, pastures, among others. Thus, although tobacco is a dicotyledonous plant, it offers clear evidence that monocotyledon genes such as sugarcane, corn, wheat and rice can confer abiotic stress tolerance on both monocotyledonous and dicotyledonous plants. ν 3/26

In recent years, with the arrival of new technologies such as genomic sequencing, microarray and other technologies (Mardis ER (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9: 387-402), gene information has increased. , to the point of patenting complete genomes (Stix G (2006) Owning the stuff of life. Sci Am 294: 76-83 and Stott M, Vaientine J (2003) Impact of gene patenting on R&D and commerce. Nat Biotechnol 21: 729-731; author reply 731). From these new approaches, a large number of genes have been identified. Thus, the patenting of plants rich in some nutrients and vitamins that have increased yield or stress tolerance, among others, has been increasingly sought.

Within stresses, abiotic types have been of particular interest for application

agriculture due to the negative effect on development and productivity in general,

• —— fear like fear of climate change. One of the most studied stress types ------

consequently with a high number of ethic patents, which is "the leading cause of water deficit or osmotic stress in plants (Cattivelli L, Rizza F, BadéeTTlFW, Mazzucotelli 'E, Mastrangelo AM, et al. (2008) Drought tolerance improvement in erop plants: An integrated view from breeding to genomics (Field Crops Research 105: 1-14).

Despite advances in the identification of genes in the most diverse genomes, there is a large group of genes throughout the genome that is not yet known for their function. These genes may be involved in many of the plant's most important pathways of stress, growth, photosynthesis, etc. response. One of the latest techniques called microarrays or DNA chips has allowed to associate function with these hitherto unknown genes. This low-cost technique lets you know the DNA sequences of hundreds of thousands of distinct genes using fluorescent molecular tags that light up when you attach a complementary strand. Thus, chips have allowed to know and identify genes involved in different plant processes, such as response to abiotic stresses, gene regulation, sucrose accumulation, pest resistance, tolerance to water scarcity, plant-pathogen interaction and so on. The mechanisms of drought tolerance in plants have similarities with other stress types (Yamaguchi-Shinozaki K, Shinozaki K (2006). Transcriptal regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology 57: 781-803 ), since the same proteins and osmoprotectants are involved in molecular responses to different types of stress. Additionally, most abiotic stresses start with a type of stress, for example, osmotic that soon triggers others. This implies that a gene or signal transduction pathway identified as responding to a specific stress may be involved in the responses of various other stress types (Yamaguchi-Shinozaki K, Shinozaki K (2006) and Zhu JK (2002). Salt and drought stress signal transduction in plants Annu Rev Plant Biol 53: 247-273).

At the cellular level, cell membranes serve as a permeable barrier to

- loss of water and certain important molecules. - However, during stress-osmotic, -a—

"availability of intercellular water is restricted," the "extracellular" concentrations of solutes leading to osmotic imbalance. This causes water to flow out of the cells, causing a decrease in cell turgency and an increase in intracellular solute concentrations. Reactive oxygen species and toxins generated during this process can also cause extensive damage to the cell.

Many patents describe different proteins that can tolerate abiotic factors such as drought and salt stress, such as dehydration-activated DNA binding elements (DREB / CBF), which make up an important family with a key role. on stress-induced signal transduction regulation (Oh S, Song S, Kim Y, Jang H, Kim S, Kim M, Kim Y, Nahm B, Kim J. (2005). Arabidopsis CBF3 / DREB1A and abf3 in transgenic Rrice increased tolerance to abiotic stress without stunting growth Plant Physiology 138: 341-351 Stockinger E Gilmour S Thomashow M. (1997) Arabidopsis Thaliana CBFl Eneodes An AP2 Domain-Containing Transeription Aetivator That Binds To The C-Repeat / DRE, A Cis-Aeting DNA Regulatory Element That Stimulates Transeription In Response To Low Temperature And Water Deficit (Proeeedings of the National Aeademy of Sciences 94: 1035-1040). CBF / DREB proteins have different domains and have been identified in different species. US7368630, US7259297 and US7253000 use these important transcriptional regulators for the production of transgenic plants that tolerate water stress.

US7368630 describes a method for using the DREB1A gene to produce a plant, tissue or plant cell line with this transcription factor, as well as dehydration-induced genes from microarray studies. US 7259297, in turn, describes transgenic plants created by introducing a gene encoding a DREB transcription factor that binds to the drought response element / crepeat (DRE) and activates the transcription of localized genes. in promoters with said DRE motif. According to this patent, DREB transcription factor overexpression activates the Rd29A, Rd29B, Rd17, Rd22, DREB1A, Cor6, Cor155, Erdl, and Kinl genes, inducing a rapid response in the plant when under stress .--- -

Similarly, US7253000 comprises a nucleic acid sequence that utilizes one of the genes belonging to the CBF group (C-repeat / dehydration-responsive element binding factor, which is another name given to DREB factors).

Other patents describe the use of various transcription factors involved in plant stress tolerance, such as W02005024028 and JP2006158402, which employ zinc finger transcription factors to confer tolerance to water stress in plants. US2008163397, in turn, used the CAAT-binding transcription factor, giving drought and cold stress tolerance, using the domain B of the transcription factor. Similarly, in US2009265813 transcription factor AP2 is used to obtain transgenic plants related to multiple traits, including enhanced root growth and obtaining drought tolerance as the end result.

The patents mentioned here reflect the biotechnological potential of the genes that

encode transcription factors, protein kinases and other proteins involved in plant responses to drought. These proteins act by directly protecting cellular components during dehydration, as well as amplifying the molecular signal of water stress perception, allowing the plant to anticipate and accelerate defense mechanisms. No document provides protection against genes related to the Scdr2 gene, the sequence of which is described in SEQID: 1 (Figure 1 A).

Despite the growth in sugarcane research, there are few examples of inventions that use sugarcane genes to produce plants with greater tolerance to abiotic stress (Affenzeller MJ, Darehshouri A, Andosch A, Lutz C, Lutz-Meindl U (2009) Salt stress-induced cell death in the unicellular green algae Micrasterias denticulata J Exp Bot 60: 939-954 and Molinari HBC, Marur CJ, Daros E, Campos MKF, Carvalho JFRP, et al. (2007) Evaluation of stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress. Physiology Plantarum 130: 218-229). And the use of sugarcane genes that give tolerance to "biotic" stresses in "Gontraponto-a" genes of other species allows the production of "cane" transgenic plants by "genes" of "sugarcane" itself. In fact, 70% of consumers support genetic modifications involving genes of their own species, in contrast to the support of 26% when dealing with genes of other species (Rommens CM (2010) Barriers). and paths to market for genetically engineered crops Plant BiotechnoIogyJournaI 8: 101-111).

Therefore, this invention relates to the direct manipulation of a DNA sequence encoding a sugarcane protein. Prior art knowledge does not allow any specific function to be associated with the target gene of this invention. This invention also describes the construction of vectors containing the sugarcane protein sequence, as well as a process of producing transgenic plants to produce a water stress tolerance, saline, higher biomass and higher photosynthesis phenotype, among others possible.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a plant production method which contains in its cells a sugarcane nucleotide sequence and the expression of this gene leads to greater tolerance to abiotic stresses to the plant in question. More broadly, the polynucleotide encoding the sugarcane protein is expressed by a plant-promoting promoter and terminator. More specifically, the present invention provides a 5 'to 3' recombinant DNA polynucleotide comprising a plant-operating promoter operably linked to a second polynucleotide encoding a cane protein operably linked to the terminator terminating transcription. of the DNA polynucleotide providing a polyadenylation site.

The invention also discloses a recombinant DNA sequence wherein the promoter is selected from the group consisting of inducible promoters, constitutive promoters, time-regulated promoters, tissue-preferred promoters, stress-specific promoters, drought-inducible promoters, inducible deficit promoters. Γ water ~, ~ is the tissue-specific promoters. —- _ ----

Also described are plant cells and plants which contain in their genome recombinant DNA molecules as described and the propagules and progeny produced therefrom. Plants include, but are not limited to, monocotyledonous or dichotiform plants, and may include sugar cane, —soya, corn, canola, rice, cotton, barley, oats, grass, wheat, pine nuts. tame, hose, guava, lemon, avocado, orange, plum, pitague, jabuticabeira, apple, peach, potato, pea, tomato, rose bush, sunflower, bean, eucalyptus, avocado, strawberry, pear, apple, guava, cocoa, lemon, passion fruit, forage palm, castor, cassava, rubber, mate, rosewood, coffee, pumpkin, watermelon, peach, pitanga and cashew.

The invention also provides a method for producing abiotic stress tolerant plants, thanks to genetic transformation with a recombinant DNA molecule expressing a sugarcane protein, as well as plants and their cells and propagules, such as seeds, containing molecules in their genome. of this recombinant DNA.

Such plants have one or more of the following properties: a higher growth rate under conditions where drought and / or salt stress would be limiting to the growth of an unprocessed plant of the same species, a higher growth rate under conditions where water would be limiting to the growth of an unprocessed plant of the same species, a higher growth rate under conditions where the increase of salts or ions in the soil and / or water would be limiting to the growth of an unprocessed plant of the same species. same species, higher percentage of plants surviving after prolonged drought or under saline stress than an unprocessed plant of the same species, a higher yield when compared to an unprocessed plant of the same species, or greater drought tolerance in compared to an unprocessed plant of the same species.

The present invention comprises the propagation of plants of this invention, for example for the purpose of generating seeds, by simply planting such seeds in the soil, or well from sprouting, as is the case with sugar cane tails. , which allows these plants to grow, for example, under stressful conditions. More specifically, this invention provides a method for producing plantains which have as one of the characteristics such as tolerance to abiotic stresses, increase in root mass yield. The invention comprises the steps of inserting in the genome of a plant cell, building a recombinant DNA molecule comprising a sugarcane gene, obtaining a transformed plant cell or transformed cells, regenerating transformed plants from plant cells and selecting plants One of the features of the invention is that the selected plants have higher tolerance to abiotic stresses selected from the group consisting of salt stress tolerance, drought tolerance and survival after the combination of abiotic stresses.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: (A) Polynucleotide sequence corresponding to SEQ ID NO: 1. (A) DNA sequence and (B) Protein deduced from the DNA fragment obtained from the sugarcane Scdr2 gene.

Figure 2: Evaluation of real-time PCR expression of the Scdr2 gene in plants subjected to water stress for 24, 72 and 120 hours. VI: SP83-5073, V2: SP90-1638, V3: SP83-2847 and V> 9/26

V4: SP86-155. Vl and V3 are tolerant sugarcane varieties, and V2 and V4 are sensitive sugarcane varieties. The asterisk indicates samples with values with statistically significant difference.

Figure 3: Effect of mannitol and NaCI on germination of plants containing SEQ ID NO: 1. Germination percentage of transgenic tobacco plants containing SEQ ID NO: 1 (Scdr2-1, Scdr2-2 and Scdr2-3) and wild plants (wt) in different concentrations of mannitol and NaCI, evaluated over 15 days. (A): control; (B): 200 mM mannitol; (C): 300 mM mannitol; (D): 100 mM NaCl and E): 175 mM NaCl.

Figure 4: Effects of saline and water stress on photosynthesis (A), Internal CO2 concentration (Ci), stomatal conductance (gs) and transpiration rate of wild plants and transgenic plants containing SEQ ID NO: 1. Plants of 30 days were subjected to 10 days of irrigation with 200 mM mannitol or 175 mM NaCl and thereafter rehydrated for 3 days with water. a- c: photosynthesis (A), d-f: internal concentration of CO2 (Ci); g-i: stomatal conductance (gs); j-l: perspiration rate (E); a, d, g and J: control treatment, b, e, h and k: 200 mM mannitol. c, f, i and I: 175 mM NaCl.

Figure 5: Water content in stressed leaves of wild plants and transgenic plants containing SEQ ID NO: 1. 30-day plants were exposed for 10 days to 200 mM mannitol or 175 mM NaCI, and after stress were irrigated with water by 3 days. In the control treatment the plants were irrigated during the whole experiment with water (n = 5).

Figure 6: Effects of drought and saline stress on dry mass of wild plants and transgenic plants containing SEQ ID NO: 1. 30-day plants were exposed for 10 days to 200 mM mannitol or 175 mM NaCI, and rehydrated for 3 days with Water.

Figure 7: Phenotype of wild (wt) tobacco plants and transgenic plants containing SEQ ID NO: 1 maintained under water and saline stress. Top Line: wt and plants of three independent events containing SEQ ID NO: 1 (Scdr2-1, Scdr2-2 and Scdr2-3) grown under normal conditions for 5 weeks. Middle line: plants irrigated with 200 mM mannitol for 10 days and irrigated with water again for 3 days. Bottom row: plants irrigated for 10 days with 175 mM NaCI and irrigated again for 3 days.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a method for producing transgenic stress-tolerant transgenic plants by introducing a gene encoding a novel sugarcane protein of previously unknown function. The gene, named Scdr2 (drought-related sugarcane), is described in the sequence SEQ ID NO: 1 (Figure IA), and the protein deduced from this DNA sequence is indicated in SEQ ID NO: 2 (Figure 1B).

The invention described herein relates to a recombinant DNA vector comprising the nucleotide sequence SEQ ID NO: 1 useful for producing transgenic plants and a method. for genetically modified plant production, which.

overexpress a nucleic acid molecule, a new sugarcane gene, which comprises SEQ ID ΝΟΓ17 involved in plant response to abiotic stresses7 and whose overexpression produces a significant improvement in plant response to these stresses. Thus, it is determined that the gene indicated in SEQ ID NO: 1 directly or indirectly controls the response of plants against these stresses.

As such, applications of the invention include, but are not limited to, improving plant yields that are tolerant to such abiotic stresses. Similarly, the invention encompasses sequences having an identity equal to or greater than 60% to SEQ ID NO: 1, but not limited to them alone.

The DNA sequence described in SEQ ID NO: 1 comprises the coding region of the Scdr2 gene that is used as part of a chimeric DNA capable of enhancing expression in plant cells.

The plant transformation vector used to introduce nucleic acid into the plant cell may be a plasmid, wherein the DNA SEQ ID NO: 1 is inserted into restriction endonuclease cleavage sites or by recombination mediated by other protein types. DNA is inserted into the cloning vector using readily known standard procedures. This usually involves the use of restriction enzymes and DNA ligases, as described, for example, by Sambrook et al (Wood EJ (1983) Molecular Cloning - A Laboratory Manual - Maniatis, T, Fritsch, Ef, Sambrook, J. Biochemical Edueation 11: 82-82). The resulting plasmid, which includes SEQ ID NO: 1, can then be used to transform a plant cell, plant or plant part by conventional methods of transformation.

For plant transformation, the preferred plasmid also includes a selection marker gene in plant transformation, although there are methods that preclude the use of such a marker gene. Commonly used plant selection markers include the kanamycin resistance gene (neomycin phosphotransferase II or nptll), the hygromycin resistance gene (hygromycin phosphotransferase or HPT), the phosphinothricin acetyl transferase (bar) gene, the 5-enolpyruvylshiquimate gene Phosphate synthase (EPSPS), or

----- acetolactate synthase (ALS): In the present invention, the selection marker is o-gene-A / pt //, which-

allows selection of kanamycin transformants. ~~ ~

The plasmid may also include a reporter gene that provides a clear indication that the genetic transformation was effected by detecting the activity of the protein encoded by the reporter gene. The most commonly used reporter genes are those that encode beta-glucuronidase (GUS), luciferase and green fluorescent protein (GFP). Reporter genes are often framed in the promoter region and in close proximity to the gene of interest to ensure they are expressed together and not separated by crossover events.

The plasmid preferably also includes promoters suitable for expression of the nucleic acid indicated in SEQ ID NO: 1 and also for expression of the selection marker gene as well as the reporter gene. The cauliflower mosaic virus 35S promoter (CaMV 35S) is commonly used for plant transformation, as is the rice actin 1 (Actl), ubiquitin 1 (Ubil) gene, the alpha gene promoter -amylase, and stress-induced gene promoters. In the present invention, the promoter used is the CaMV 35S promoter, but other promoters may also be used. In the plasmid, the DNA described in SEQ ID NO: 1 may be under the control of a constitutive promoter or may be under the control of the same gene promoter or a different promoter. For plant transformation, the plasmid preferably also includes a terminator-encoding nucleic acid molecule, such as the non-coding 3 'region of the genes encoding an actin protease inhibitor, cauliflower mosaic virus, or nopaline (NOS). In the present invention, the plasmid includes the nopaline synthase terminator (NOS).

The plasmid is preferably a binary plant transformation vector in which the genes of interest are inserted within the edges of the T-DNA. Examples of such plant transformation vectors that may be used in the present invention are vectors obtained from commercial sources, such as the pCambia, pBH21 or pGreenll series which contain a

------ 0RequestRK2fromreplication0r0-genemarcadrr-n ^ and o—

Synthetic "nopaline terminator" (NÜS), constitutive promoter, and a polyadenylation "3-NOS" siall.

For the transformation of plants, any suitable method for the transformation of

Monocotyledons or dicotyledons can be used, such as Agrobacterium-mediated transformation or particle bombardment (also known as biobalistic transformation). In Agrobacterium-mediated transformation, plant cells are contacted with an inoculum of bacteria transformed with the DNA-containing plasmid indicated in SEQ ID NO: 1 of the invention, for example by inoculating plant cells with a bacterial suspension. transformed. Bacteria of the genus Agrobacterium that can be used to transform plant cells include species of Agrobacterium rhizogenes, Agrobacterium tumefaciens, preferably A. tumefaciens LBA4404, EHA105 or GV301 strains. Agrobacterium spp. are transformed with the plasmid by known conventional methods.

In turn, using a leaf disc transformation protocol, A. tumefaciens bacteria with the DNA indicated in SEQ ID NO: 1 cloned into the binary vector are grown in a growth medium in the presence of antibiotics such as kanamycin, to select the bacterial cells that have the binary plasmid. Wild tobacco leaves are then surface sterilized, cut into small disks and incubated in a suspension of A. tumefaciens for a suitable time. Different fabrics can be used for processing, such as leaves, stalk, flower among others. After incubation with A. tumefaciens the infected leaves are transferred to the culture medium in vitro for a certain period of time. Selection of transgenic plants is initiated by placing infected plants in the in vitro selection medium with antibiotic. After the development of rooted seedlings occurs the transfer to soil and growth in growth chamber.

Additionally, the present invention relates to the production of plasmid-transformed transgenic plants containing the sequence indicated in SEQ ID NO: 1, increasing their tolerance to abiotic stresses such as drought and high salinity.

Another approach to increasing expression levels of SEQ ID NO: 1 may be the production of genetically modified plants that overexpress genes that induce endogenous gene promoter activity, such as genes encoding transcription factors. - - - = -

Thus, the invention describes the method for producing transgenic plants which contains in their cells a chimeric gene capable of expression in plant cells, as well as subsequent generations comprising a DNA sequence of SEQ ID NO: 1 and DNA sequences that allow expression of said protein in plant cells.

Transgenic plants according to the invention include, without limitation, cereals such as wheat, barley, maize, rice, oats, fodder grasses, peat, other monocotyledons such as sugar cane, miscanthus, or any species of other foods such as beans, soybeans, peas, tomatoes, rapeseed, as well as other species of economic interest, such as tobacco, oranges, etc. EXAMPLES

Example 1: EVALUATION OF THE SUGR2 GENE EXPRESSION LEVEL OF SUGAR

To evaluate the pattern of expression of the dry gene Scdr2, a quantitative Real-time PCR was performed as described by Rocha et al (Rocha FR, Papini-TerzI FS, Nishiyama MY, Vencio RZN, Vicentini R, et al. (2007). Signal Bmc Genomics 8) using sugarcane leaves with higher drought tolerance (SP83-5073 and SP83-2847) and two with higher drought sensitivity (SP86-155 and SP90- 1638) maintained under irrigation or rainfed (with irrigation suspension, equivalent to drought stress). As reference gene the sugarcane GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene was used.

_A ^ pressure_sj) b_s_ecaJsequjeiro) JoLdeterminad -

--- Sehmittgen TD (2001) Analysis of relative gene expression data using quantitative real-time "

PCR and the 2 (-Delta Delta C (T)) Method. Methods 25: 402-408), Using "non-stressed" (irrigated) samples as a control. Statistical significance of relative gene expression differences was determined by assuming a log-normal model that calculates the probability Pr (sample> reference>) and Pr (sample <reference) for induced and repressed genes, respectively. Expression Profile was considered valid when P> 0.95. For each treatment, leaves from six plants were used.

The analysis result showed that Scdr2 gene expression was strongly induced after 24 hours of stress in drought tolerant varieties, and late induced (72 hours) in sensitive varieties, which could explain the contrasting drought tolerance levels (Figure 2). . These data suggested that the Scdr2 gene could be involved in sugarcane drought tolerance.

Example 2: CLONING OF SEQ ID NO: 1 SUGAR CANE AND PRODUCTION OF A RECOMBINANT CASSETTE.

One clone with sugarcane Scdr2 gene sequence was obtained from the Brazilian Clone Collection Center (BCCCENTER, Jaboticabal, Brazil). From this DNA the complete coding sequence, indicated in SEQ ID NO: 1 (Figure 1), was PCR amplified using specific 5-GGATCCCTCATCGCCAGCTCCCAT-3- 'and 5'-TCTAGACCTGTGCAGTGTCGGATTATTC-3' oligonucleotides, and cloned into pGEMT- Easy (Promega, USA).

Subsequently, the coding region of the Scdr2 gene containing SEQ ID NO: 1 was removed with the enzymes BamHI and Xbal and inserted into the pRT104 vector digested with the same enzymes. The expression cassette was released by HindIII digestion and cloned into the pCAMBIA 2301 (Cambia, Australia) expression vector, also digested with the same enzyme. The resulting final recombinant construct (pCAMBIA2301 :: Sci / r2) was introduced into Agrobacterium tumefaciens strain GV3101 (CLONTECH, USA).

Example 3: GENETICALLY MODIFIED PLANT PRODUCTION

Wild tobacco seeds (Nicotiana tabacum, var. SRl) were germinated in a petri dish containing Murashige-Skoog (MS) medium with 0.9% (w / v) agar. The seedlings were transplanted to soil, composed of Baccto (Mjchigan Peat Co., USA) and = sand (4: 1, v / v) in 325 ml pots. When the plants developed between 10 and 12 leaves, they were transplanted to 1 L pots containing the same mixture mentioned above.

The plants were grown in a growth chamber with 16 / 8h light / dark (300-400 μιηοΙ photons ItT2S "1) photoperiod at 25 ° C and 75-80% relative humidity. For seed production, plants were induced to flowering by length of time of exposure to light = -

Wild tobacco leaves were sterilized on their surface, cut into small discs and incubated in a suspension of A. tumefaciens for 5 to 20 min. After this, the infected leaves were transferred to MS medium (supplemented with 2 mg L1 benzyl adenine and 0.1 mg L1 naphthylacetic acid) for 3 days.

Selection of transgenic plants was initiated by placing infected plants in the selection medium (MS, 2 mg / L "1 benzyladenine, 0.1 mg / L1 naphthylacetic acid and 100 mg / L" 1 kanamycin, or 30 mg / L "1 hygromycin and 500 mg / L "1 carbenicillin). The explants that then developed were transferred to MS medium with 0.1 mg / l "l 3-indole acetic acid, 100 mg / l" 1 kanamycin, or 30 mg / l "1 hygromycin, and 500 mg / l" 1 carbenicillin while rooted plants were transferred to soil and grown in a 16 / 8h light / dark (300-400 μηποΙ photon m-2 s "1) photoperiod at 25 ° C and 75-80% relative humidity .

Example 4: ANALYSIS OF THE PRESENCE OF SEQ ID NO: 1 IN GENETICALLY MODIFIED PLANTS

The initial characterization of rooted and transferred plarites was first performed by the histochemical assay using X-Gluc as substrate for detection of beta-glucuronidase gene, also inserted in the expression cassette.

It was observed that a large number of plants had a strong blue color, which were used to confirm the integration of the transgene containing the Scdr2 gene indicated in SEQ ID NO: 1 by PCR using genomic DNA.o.molde ._ ^ ------

Then total DNA was extracted from leaf samples from wild plants and transgenic plants. For this, about 50 - 150 mg of leaves were collected and quickly transferred to a mortar containing liquid nitrogen. After evaporation in nitrogen, the material was sprayed to a very fine powder to which was added 4 mL of extraction buffer. Immediately the solution was transferred to sterile 1.5 ml plastic tubes which were kept in a water bath at 65 ° C for 30 minutes with gentle agitation every 10 minutes.

Immediately thereafter, 10 microliters of Proteinase K was added to the tubes and incubated again in a 65 ° C water bath for 30 minutes, with gentle shaking every 10 minutes. The tubes were then centrifuged in a microcentrifuge at 10,000 rpm for 10 minutes. The supernatant was then transferred to a new 1.5 ml plastic tube and, after reaching room temperature, the first DNA extraction was performed using 1: 1 phenol-chloroform (1: 1). part of the supernatant: 1 part phenol-chloroform).

In sequence, the tube was gently inverted several times to achieve a homogeneous emulsion and the samples were centrifuged at 10,000 rpm for 10 minutes. Immediately after the tubes were carefully removed from the microcentrifuge, the upper (aqueous) phase was transferred to a new 1.5 mL tube, avoiding contaminants from the lower phase. Immediately thereafter, 100 mg / ml RNAse (1: 100) was added and the mixture was incubated at 37 ° C for 30 minutes.

In the last phase of extraction, the same volume of chlorophyll solution (24

parts chloroform: 1 part isoamyl alcohol) and the mixture was stirred gently for 5 minutes. The tubes were centrifuged at 10,000 rpm for 10 minutes and the upper phase was transferred to a new container.

Finally, the same volume of chlorophyll solution was added and the mixture was stirred gently. The tubes were then centrifuged at 10,000 rpm for 10 minutes and the upper phase was transferred to a new tube. To this upper aqueous phase was added 1 volume of isopropyl alcohol and the solution was then homogenized until a DNA precipitate formed. Precipitated DNA was washed in the tube itself in 70% alcohol three or three times and 100 µl of ultra pure water was added. Genomic DNA was stored at 4 ° C.

The presence of SEQ ID NO: 1 in genomic DNA extracted from transgenic plants of

tobacco was confirmed by polymerase chain reaction (PCR). Initially, 1μΙ_ DNA, 1.5μΙ_ 4 dNTP (IOmM), 1.4μί were used. MgCl 2 (25mM), μμΙ. of Primer sense (10μΜ), ΙμΙ

____ AntLsense Primer (10 ^ M), 2.5 ^ L Taq Buffer 10x, 0.3 µL τaq Polymerase (Ferments)) 16.3μL-de

MiIIiQ water and this reaction was subjected to the following amplification program: 3 minutes at 94 ° C plus 30 cycles of 30 seconds at 94 ° C, 30 seconds at a temperature of 54 ° C, 2 minutes at 72 ° C and 7 minutes at 72 ° C.

The product of this reaction was visualized on 0.8% agarose gel in TAE containing ethidium bromide under ultraviolet light illumination. Among 12 allegedly transgenic plant samples, the presence of SEQ ID NO: 1 was confirmed in 8. These plants were used in subsequent assays to observe the effects of the Scdr2 gene indicated in SEQID NO: 1 on the characteristics of tobacco plants. Example 5: GERMINATION ANALYSIS OF GENETICALLY MODIFIED PLANT SEEDS CONTAINING SEQ ID NO: 1 UNDER CONDITIONS OF WATER STRESS

To determine the effects of water stress on germination, seeds of wild type and transgenic tobacco plants identified in Example 4 were used.

The seeds were superficially sterilized with 70% alcohol for 1 min, incubated in 2% NaCIO for 30 min and washed five to six times in sterile distilled water. Seeds were sown in Petri dishes (30 seeds per plate) containing solid Murashige-Skoog (MS) medium, pH 5.8, in a 23 ° C chamber with 16/8 h light / dark photoperiod (300-400 mmol photons m "2 s" 1).

Then, the seeds that were transformed into JreseventsJndependeVites (Scdr2-1, __ _ Scdr2-2 and-Scdr2-3) were used to evaluate the percentage of germination in mannitol-containing culture by verifying the response to hydrical stress. To test the effect of drought, mannitol was used in the culture medium at concentrations of 0, 200, 300 and 400 mM. The number of germinated seeds was observed daily for 15 days and germination was defined as the appearance of the hypocotyl.

In the control condition without mannitol, seeds from wild plants and plants containing SEQiD-NOTl showed similar germination percentage (Figure 3A). Water stress inhibits or reduces germination in tobacco plants. In medium containing 200 mM mannitol the seeds of plants containing SEQ ID NO: 1 showed a faster germination compared to wild seeds (Figure 3B). Under conditions of higher water stress (300 mM), wild plant germination, but transgenic plants reached approximately 60% (Figure 3C). Therefore, plants with the Scdr2 gene indicated in SEQ ID NO: 1 showed higher tolerance to water stress.

In numerous cultivated plants, germination and initial seedling growth are

the most sensitive phases to abiotic stresses. For example, some environmental factors such as low temperatures, high salt concentrations or water deficit in the environment significantly retard germination and reduce the rate of germination events. Low seed germination rate can result in uneven soil establishment and reduced yield. A desirable plant characteristic for the production of commercial cultivars under field conditions is the ability of the seed to germinate rapidly and evenly under stress conditions.

Transgenic plants containing SEQ ID NO: 1 germinated 40% more than wild plants at high stress concentrations. The performance of transgenic tobacco seeds carrying the gene of interest was better than wild plants under water stress conditions, indicating that the study gene plays a protective role in the early stages of plant development.

Example 6: ANALYSIS OF SALIN STRESS GERMINATION

... To determine the effects of water-saline stress on germination forr used seeds of transgenic wild-type and transgenic tobacco plants identified in example 4.

The seeds were superficially sterilized with alcohol, 70% for 1 min, incubated in 2% NaCIO for 30 min and washed five to six times in sterile distilled water. The seeds were sown in Petri dishes (30 seeds per plate) containing solid Murashige-Skoog (MS) medium, 1dH3,8 ~ in a 23 ° C chamber with 16/8 h light / dark photoperiod (300-400 mmol photons). rrr2 s "1) and to test the effect of salt stress NaCl (0, 100, 175 mM) was used in the culture medium. The number of germinated seeds was observed daily for 15 days, and germination was defined as the emergence of hypocotyl.

When seeds were plated at 100 mM NaCI transgenic plants containing SEQ ID NO: 1 germinated much faster than wild plants. 50% of transgenic plant germination was reached on the seventh day, while wild plants reached 50% only on the eleventh day 11 (Figure 3D). However, using a more severe stress concentration (175 mM NaCl), germination was completely inhibited in wild seeds, but only moderately in plants containing SEQ ID NO: 1, which reached 20% germination (Figure 3E). . The results demonstrate that the Scdr2 gene, indicated in SEQ ID NO: 1 in transgenic plants, improves germination under salt stress.

Different responses to abiotic stresses are the result of cooperative interactions between the various biochemical, physiological and morphological character components. These interactions may vary between species and even varieties, as noted for water stress. Plants containing SEQ ID NO: 1 showed a high level of salt stress tolerance.

As mentioned earlier, germination and seedling growth are very sensitive phases to stressful conditions. In turn, soil salinity is a global problem, which not only precludes the use of large areas for agriculture, but may also reduce productivity in certain agricultural areas due to saline increase due to irrigation with water in the propriety by the present disclosed salt content. _ „

Under high salinity conditions, while wild plants showed inhibition of germination, transgenic plants containing SEQ ID NO: 1 presented higher germination percentage. Accordingly, this invention shows that SEQ ID NO: 1 is useful for producing genetically modified plants with greater salinity tolerance.

Example 7: EVALUATION OF PHOTOSYNTHETIC PARAMETERS OF --TRANSGENIC PLANTS CONTAINING SEQ ID NO: 1 SUBMITTED TO WATER STRESS.

To evaluate the response to water stress, wild plants and transgenic plants containing SEQ ID NO: 1 were germinated for 16 days and then transferred to Plantmax HT (Eucatex, Brazil) pots in a growth chamber at 22 ° C and 18 ° C. hours of light. The plants were irrigated daily with 70 ml of water for 4 weeks before stress began.

For water stress, five plants per treatment and genotype (wild and transgenic) were used, which were irrigated with 100 ml of 200 mM mannitol for 10 days and then irrigated again with water for three days, as described by Zhang et al. al. (Zhang XX, Liu SK, Takano T (2008) Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing salt, drought, oxidation and cold tolerance. Plant Molecular Biology 68: 131-143).

To estimate the stomatal conductance of CO2 (gs), transpiration rate (E), photosynthesis (A) and internal concentration of CO2 (Ci), under control conditions and under water stress, completely expanded leaves were used in the same position of the plants. tobacco.

Three independent events of transgenic plants containing SEQ ID NO: _1 and wild plants were evaluated. Measurements were performed with IRGA equipment (LCpro +; ADC bioscientific, United Kingdom) at a CO2 concentration of 360 μί L "1, light intensity of 1000 μιηοΙ Itf2S" 1, gas flow 200 ml_ min "1 and temperature within 25 ° C chamber (Guo Q, Zhang J, Gao Q, Xing S, Li F, et al. (2008) Drought tolerance through overexpression of monoubiquitin in transgenic tobacco.

However, the physiological variables analyzed were negatively affected by water stress in both wild and transgenic plants containing SEQ ID NO: 1. However, transgenic plants performed better in the analyzes. physiological tests performed. The photosynthesis rate (A) in transgenic plants containing SEQ ID NO: 1, submitted to water stress, showed a fast recovery when they were. plants were rehydrated for 3 days, unlike wild plants that failed to recover (day 13 in Figure 4B).

Similarly, both gs (Figure 4H) and E (Figure 4K) were quickly recovered during rehydration after water stress. Ci showed similar values under normal conditions, whereas under stress conditions plants containing SEQ ID NO:

1 performed better than wild plants (Figure 4E). In summary, a clear difference was observed between transgenic and wild plants in all photosynthetic parameters evaluated under drought conditions.

Crops usually experience water scarcity at some point.

throughout its life cycle and, depending on the duration and intensity of water stress, significant losses in productivity may occur. Important parameters of plant physiology when subjected to water stress were less affected in those containing SEQ ID NO: 1 compared to wild plants. This indicates that the present invention contributes to the production of genetically modified plants with better performance under water scarcity conditions.

Example 8: EVALUATION OF PHOTOSYTHETIC PARAMETERS OF TRANSGENIC PLANTS CONTAINING SEQ ID NO: 1 SUBMITTED TO SALINE STRESS

To evaluate the effect of SEQ ID NO: 1 on saline stress conditions, wild plants and transgenic plants containing SEQ ID NO: 1 were germinated for 16 days and then transferred to Plantmax HT pots (Eucatex, Brazil) in a growth chamber. at 22 ° C and 18 hours of light. The plants were irrigated daily with 100 ml of water for 4 weeks before stress began.

To evaluate the effect_ of stress_sal ^ -

____ regularly irrigated with water for five weeks (control treatment), while a group

of plants was irrigated with 175 mM NaCl for -10 days and subsequently rehydrated for three days with water (control plants maintained irrigation for the duration of the experiment).

Three independent events of transgenic plants containing SEQ ID NO: 1 were evaluated in terms of stomatal conductance (gs), transpiration rate (E), photosynthesis (A), and internal CO2 concentration (Ci) under controlled conditions and under controlled conditions. saline stress.

The evaluation of photosynthesis rate (A) in transgenic plants containing SEQ ID NO: 1 was reduced in response to saline stress, showing a rapid recovery when plants were rehydrated for three days, while wild plants did not recover ( Figure 4C). In turn, both gels (Figure 41) and E (Figure 4L) were rapidly recovered during rehydration following salt stress. Ci showed similar values under normal conditions, although under stress conditions plants containing SEQ ID NO: 1 performed better than wild plants (Figure 4F). In summary, it was observed that plants containing SEQ ID NO: 1 performed better than wild plants in all photosynthetic parameters evaluated during salt stress.

Example 9: EVALUATION OF THE WATER CONTENT OF TRANSGENIC PLANTS CONTAINING SEQ ID NO: 1 SUBMITTED TO WATER STRESS.

Wild and transgenic tobacco seeds containing SEQ ID NO: 1 were germinated for 16 days in Petri dishes containing MS medium and pH 5.8. The seedlings were then transferred to 500 ml pots containing Plantmax HT (Eucatex, Brazil) for 3 weeks kept in a growth chamber at 23 ° C with 16/8 h light / dark photoperiod and fertilized weekly with nutrient solution (EPPQ, Brazil). . Each plant was irrigated with 100 ml of a 200 mM mannitol solution for 10 days and then irrigated for three days --- with pure water for recovery. Γ "

To estimate leaf water content, plant samples were incubated at 80 ° C for 24 hours to assess their dry weight. Leaf water content was calculated as 100x (FW-DW) / (FW), with FW being fresh mass and DW being dry weight. Under water stress the leaf-water content was dramatically reduced in wild plants (Figure 5). In contrast, transgenic plants containing SEQ ID NO: 1 were able to keep their water content almost unchanged, indicating that SEQ ID NO : 1 confers_a_capacity._bigger- to maintain turgence under water stress conditions.

Example 10: EVALUATION OF TRANSGENIC PLANT WATER CONTENT

CONTAINING SEQ ID NO: 1 SUBMITTED TO SALINE STRESS.

Wild and transgenic tobacco seeds containing SEQ ID NO: 1 were germinated for 16 days in Petri dishes containing MS medium and pH 5.8. The seedlings were transferred to 500 ml pots containing Plantmax HT (Eucatex, Brazil) for 3 weeks and kept in a growth chamber at 23 ° C with 16/8 h light / dark photoperiod and fertilized weekly with nutrient solution (EPPQ, Brazil). . Each plant was irrigated with 100 ml of a 175 mM NaCl solution for 10 days and then irrigated for three days with pure water for recovery.

To estimate leaf water content, plant samples were incubated at 80 ° C for 24 hours to evaluate their dry weight. Leaf water content was calculated as 100x (FW-DW) / (FW), with FW being fresh mass and DW being dry weight.

Saline stress decreased water content in wild plants, whereas transgenic plants containing SEQ ID NO: 1 were able to keep leaf water content almost unchanged (Figure 5), indicating that plants that overexpress Scdr2 are capable of to maintain turgidity under stressful conditions.

Example 11: EVALUATION OF THE EFFECT OF SEQ ID NO: 1 ON THE DRY MASS OF WATER-STRESSED PLANTS. ____________________________

- Wild tobacco plants and transgenic plants containing SEQ ID NO: 1 were germinated for 16 days and then transferred to containers containing Plantmax HT (Eucatex, Brazil). The plants were deposited in a growth chamber at 22 ° C with 18 hours of light and irrigated daily with 100 ml of water for 4 weeks before stress began. For water stress the seedlings were irrigated with 100 ml of 200 mM mannitol,

for 10 days, and then were irrigated again with water for three days as described by

__> _____—

Zhang et al JZhang-XX, ^ iu- SK, -Takano-T- (2008) Two ^ inhibitors from

Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance. Plant Molecular Biology 68: 131-143).

Plant samples were collected and maintained at 80 ° C for 24 hours for root and shoot dry matter evaluation, as described by Guo et al. (Guo Q, Zhang J, Gao Q, Xing S, Li F, et al. (2008) Drought tolerance through overexpression of monoubiquitin in transgenic tobacco. Journal of Plant Physiology 165: 1745-1755).

A reduction in the mass of wild and transgenic plants was observed when

submitted to drought compared to the control treatment. However, at the end of the water stress treatment, transgenic plants containing SEQ ID NO: 1 had more dry mass than wild plants (Figure 6).

Example 12: EVALUATION OF THE EFFECT OF SEQ ID NO: 1 ON DRY PASTA OF SALINE STRESS PLANTS.

Wild tobacco plants and transgenic plants containing SEQ ID NO: 1 were germinated for 16 days and then transferred to Plantmax HT pots (Eucatex, Brazil), located in a growth chamber at 22 ° C with 18 hours of light and were irrigated daily with 100 ml of water for 4 weeks before stress begins. For salt stress the plants were irrigated with 100 ml of 175 mM NaCl for 10 days and then irrigated again with water for three days as described by Zhang et al. (Zhang XX, Liu SK, Takano T (2008) Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSbJcreasing the salt, drought / oxidation of cpld tolerance. Plant Molecular Biology 68: 131-143) .------- ----- ~

Transformed plant samples were collected and maintained at 80 ° C for 24 hours to evaluate root and shoot dry mass, as described by Guo et al. (Guo Q, Zhang J, Gao Q, Xing S, Li F, et al. (2008) Drought tolerance through overexpression of monoubiquitin in transgenic tobacco. Journal of Plant Physiology 165: 1745-1755).

Stress - saline - reduces - the dry mass of wild and transgenic plants has been observed. However, the presence of SEQ ID NO: 1 in transgenic plants was associated with a higher dry matter accumulation capacity under saline stress conditions (Figure 6).

Example 13: VISUAL INSPECTION OF THE EFFECT OF WATER AND SALINE STRESS ON WILD TOBACCO PLANTS AND TRANSGENIC PLANTS CONTAINING SEQ ID NO: í.

To evaluate the response to water and saline stresses, wild plants and transgenic plants containing SEQ ID NO: 1 were germinated for 16 days and. then transferred to pots containing Plantmax HT (Eucatex, Brazil) in a growth chamber at 22 ° C with 18 hours of light and irrigated daily with 100 ml of water for 4 weeks before stress began. To assess the effect of water stress the seedlings were irrigated with 100 ml 200 mM mannitol for 10 days and then irrigated again with water for three days as described by Zhang et al. (Zhang XX, Liu SK, Takano T (2008) Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing salt, drought, oxidation and cold tolerance. Plant Molecular Biology 68: 131-143) and to evaluate the effect of Saline stress plants were irrigated with 175 mM NaCl for 10 days and subsequently rehydrated for three days with water. In the control treatment the plants were irrigated during the experiment time.

Under control conditions, all plants had similar results (Figure 7), while under water and saline stress the leaves of wild plants withered. In turn, transgenic plants containing SEQ ID NO: 1 exhibited a phenotype virtually similar to plants maintained without stress (figureJ7j ^ __________ __________

----- TheseLmesmas.plants.were used in the analyzes shown in examples 7 and 8 and are

in agreement with the best performance of the plants in relation to the photosynthetic parameters. Similarly, this phenotype observed in Figure 7 is also a reflection of the higher water contents shown in examples 9 and 10, as well as the higher dry mass shown in examples 11 and 12.

Claims (39)

Method for the production of genetically modified plants, comprising the following steps: (a) production of a vector comprising the sequence described in SEQ ID NO: 1; b) Production of a vector wherein the sequence described in SEQ ID NO: 1 is operably linked to a functional promoter in plants; c) Production of a vector in which the sequence described in SEQ ID NO: 1 is operatively linked to a functional terminator in plants; d) Insertion of said vector comprising the sequence described in SEQID NO: 1, the promoter and the terminator into the plant cell genome, plant or plant parts. A method for producing genetically modified plants, comprising the following steps: (a) producing a vector comprising at least 60% identity with the sequence described in SEQ ID NO: 1; (b) producing a vector in which the sequence of at least 60% identity to the sequence described in SEQ ID NO: 1 is operatively linked to a functional promoter in plants; c) Production of a vector in which the sequence of at least 60% identity to the sequence described in SEQ ID NO: 1 is operatively linked to a functional terminator in plants; d) Insertion of said vector comprising the sequence of at least 60% identity to that described in SEQ ID NO: 1, the promoter and terminator in the plant cell genome, plant or plant parts. Method according to claim 1, characterized in that genetically modified plants have in their genome the gene sequence SEQ ID NO: 1. Method according to claim 2, characterized in that genetically modified plants have in their genome a gene sequence of at least 60% identity with SEQID NO: 1. Method according to claim 1, characterized in that the gene sequence SEQ ID NO: 1 is obtained from sugar cane. Method according to Claims 1 and 2, characterized in that it can be applied to monocotyledonias. Method according to Claims 1 and 2, characterized in that it can be applied to dicotyledons. Method according to Claims 1 and 2, characterized in that it is applied to tobacco. Method according to Claims 1 and 2, characterized in that it is applied to sugar cane, soybean, corn, rice and wheat. Method according to claims 1 and 2, characterized in that it allows the integration of the vector into the plant cell genome. Method according to Claims 1 and 2, characterized in that it modulates the tolerance of the plant to environmental stresses. Method according to claim 11, characterized in that the environmental stress is preferably dry. Method according to claim 11, characterized in that the environmental stress is preferably salinity. 14. Recombinant DNA vector, characterized in that it comprises the gene sequence described in SEQ ID NO: 1. Vector according to claim 14, characterized in that it comprises a polynucleotide sequence responsible for producing a polypeptide described in SEQ ID NO: 2. 16. Recombinant DNA vector, characterized in that it comprises a gene sequence with at least 60% identity to the sequence described in SEQ ID NO: 1. Vector according to claim 16, characterized in that it comprises a polynucleotide sequence responsible for producing a polypeptide with at least 60% identity to SEQ ID NO: 2. Vector according to claims 14 and 16, characterized in that it is an expression vector or a cloning vector. Vector according to claims 14 and 16, characterized in that it can be a plasmid. Vector according to claims 14 and 16, characterized in that the genes of interest are inserted between the edges of the T-DNA of a binary vector. Vector according to claims 14 and 16, characterized in that it contains a promoter operably linked to the gene sequence of interest. Vector according to claim 21, characterized in that the promoter is preferably the CaMV 35 S promoter of the cauliflower mosaic virus. Vector according to claims 14 and 16, characterized in that it contains a terminator operably linked to the gene sequence of interest. Vector according to claim 23, characterized in that the nucleic acid encoding the terminator is preferably nopaline synthase (NOS). Vector according to claims 14 and 16, characterized in that it may contain a marker gene. Vector according to claim 25, characterized in that the marker gene is preferably the NptIL gene. Vector according to claims 14 and 16, characterized in that it may contain a reporter gene. Vector according to claims 14 and 16, characterized in that it is capable of transferring DNA to plant cells, plants or parts of plants. Use of the method described in claims 1 to 13 characterized in that it is applied for the production of plant cell, plant or plant parts. Use of the method described in claims 1 to 13 characterized in that it is applied to seed production from plants containing the sequence described in SEQ ID NO: 1. Use of the method described in claims 1 to 13 characterized in that it is applied for seed production from plants containing gene sequence with at least 60% identity to that described in SEQ ID NO: 1. Use of the method described in claims 1 to 13 for applying to parts of plants used for the purpose of asexual reproduction from plants containing the sequence described in SEQ ID NO: 1. Use of the method described in claims 1 to 13 characterized in that it is applied for the production of parts of plants used for the purpose of asexual reproduction from plants containing at least 60% identity sequence to that described in SEQID NO. : l. Use of the method described in claims 1 to 13 for application to plant progeny containing the sequence described in SEQ ID NO: 1. Use of the method described in claims 1 to 13 for application to plant progeny containing gene sequence of at least 60% identity to that described in SEQID NO: 1-. Use of the vector described in claims 14 to 28 characterized in that it contains the gene sequence described in SEQID NO: 1. Use of the vector described in claims 14 to 28 characterized in that it contains a gene sequence of at least 60% identity to that described in SEQ ID NO: 1. 38. Use of the gene sequence described in SEQ ID NO: 1 characterized in that it can be applied to monocotyledons or dicotyledons. 39. Use of gene sequences that are at least 60% identical with the gene sequence described in SEQ ID NO: 1, characterized in that it can be applied to monocotyledons or dicotyledons.