BRPI1009965B1 - METHOD FOR PRODUCTION OF ENVIRONMENTAL STRESS TOLERANT PLANTS, THEIR USES AND RECOMBINANT DNA VECTOR - Google Patents

Abstract

METHOD FOR PRODUCTION OF ENVIRONMENTAL STRESS TOLERANT PLANTS, THEIR USES AND RECOMBINANT DNA VECTOR. Plants are influenced by a large number of biotic and abiotic environmental factors and recurrent 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 method of producing plants that contain in their cells a sequence of sugarcane nucleotides and the overexpression of this gene leads the plant in question to greater tolerance to abiotic stresses. More broadly, a polynucleotide encoding sugar cane protein, which is expressed by a promoter and terminator that function in plants, is described. It is verified that this gene confers tolerance to different abiotic stresses.

Description

FIELD OF THE INVENTION

Plants are influenced by a large number of environmental, biotic and abiotic factors and recurrent abiotic stresses, such as drought, salinity, temperature, pollution, radiation, etc., are more severe and affect all plant functions, resulting in reduced growth and growth. of productivity. It is estimated that this type of stress generally affects physiological, biochemical and morphological levels, and can reduce productivity by 50% and in some cases up to 70%. This has led to efforts to understand 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 the production of transgenic plants tolerant to environmental stresses thanks to the introduction of a vector that comprises a gene that encodes a new sugarcane protein, of unknown function, in addition to the construction of the vector and its uses.

FUNDAMENTALS 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 limiting water conditions. In recent decades, there has been a strong investment in the production of transgenic plants tolerant to stress, among which drought and saline stress stand out. 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 the production of tolerant plants, 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 Biotechniques 10: 294-300; Rodenburg KW, de Groot MJ, Schilperoort RA, Hooykaas PJ (1989) Single-stranded DNA used as an efficient new vehicle for transformation of plan t protoplasts. Plant Mol Biol 13: 711-719 and Ballas N, Zakai N, Loyter A (1987) Transient expression of the pCaMVCAT plasmid in plant protoplasts following transformation with polyethyleneglycol. Exp Cell Res 170: 228-234) and 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 plant transformation and research has 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 due to 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 tool for gene transformation due to the ease of transformation and rapid obtaining of transformed generations. In this way, genes from different species can be initially evaluated in tobacco, due to the ease and speed in the production and analysis of transgenic plants. This has occurred with genes from cereals, vegetables, ornamental and medicinal plants, fruit trees, pastures, among others. Thus, although tobacco is a dicotyledonous plant, it offers clear evidence that genes from monocots, such as sugarcane, corn, wheat, and rice, can confer tolerance to abiotic stresses in both monocots and dicots.

In recent years, with the arrival of new technologies such as genomic sequencing, microarrays and other technologies (Mardis ER (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9: 387-402) information about genes has increased. , to the point of patenting whole genomes (Stix G (2006) Owning the stuff of life. Sci Am 294:76-83 and Stott M, Valentine 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. In this way, the patenting of plants rich in some nutrients and vitamins, which present an increase in production or tolerance to stress, among others, has been increasingly sought.

Among the stresses, the abiotic types have been of particular interest for application in agriculture due to the negative effect on development and productivity in general, as well as the fear of climate change. One of the most studied types of stress and consequently with a high number of patents is drought, which is the main cause of water deficit or osmotic stress in plants (Cattivelli L, Rizza F, Badeck FW, Mazzucotelli E, Mastrangelo AM, et al. (2008) Drought tolerance improvement in crop 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 whose function is still unknown. These genes may be involved in many of the most important plant response pathways to stress, growth, photosynthesis, etc. One of the most recent techniques called microarrays or DNA chips has made it possible to associate function with these hitherto unknown genes. This low-cost technique makes it possible to know the DNA sequences of hundreds of thousands of distinct genes using fluorescent molecular tags that light up when a complementary strand is ligated. In this way, chips have made it possible to know and identify genes involved in different plant processes, such as response to abiotic stresses, gene regulation, sucrose accumulation, as well as resistance to pests, tolerance to water scarcity, plant-pathogen interaction, etc.

The mechanisms of drought tolerance in plants have similarities with other types of stresses (Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional 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 one type of stress, eg osmotic, which soon trigger the others. This implies that a gene or signal transduction pathway identified as a response to a specific stress may be involved in responses to several other types of stress (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 the loss of water and some important molecules. However, during osmotic stress, the availability of intercellular water is restricted, which alters the extracellular concentrations of solutes, leading to an osmotic imbalance. In this way, water flows out of the cells, causing a decrease in cell turgor and an increase in intracellular concentrations of solutes. The reactive oxygen species and toxins generated during this process can also cause extensive damage to the cell.

There are many patents that describe different proteins capable of conferring tolerance to abiotic factors such as drought and salt stress, such as the DNA binding elements activated in response to dehydration (DREB/CBF), which make up an important family with a key role. in the regulation of stress-induced signal transduction (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 CBF1 Encodes An AP2 Domain-Containing Transcription Activator That Binds To The C-Repeat/ DRE, A Cis-Acting DNA Regulatory Element That Stimulates Transcription In Response To Low Temperature And Water Deficit. Proceedings of the National Academy of Sciences. 94:1035-1040). CBF/DREB proteins have different domains and have been identified in different species. Documents US7368630, US7259297 and US7253000 use these important transcriptional regulators for the production of transgenic plants tolerant to water stress.

US7368630 patent describes a method for using the DREB1A gene to produce a plant cell line, tissue or plant with this transcription factor, as well as dehydration-induced genes from microarray studies. In turn, document US7259297 describes transgenic plants created by introducing a gene that encodes a DREB transcription factor that binds to the drought response element DRE/CRT (Drought Response Element/Crepeat) and activates the transcription of localized genes. in promoters with said DRE motif. According to this patent, the overexpression of the transcription factor DREB activates the genes Rd29A, Rd29B, Rdl7, Rd22, DREB1A, Cor6, Corl5a, Erdl and Kinl, inducing a rapid response in the plant when it is subjected to stress.

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

Other patents describe the use of various transcription factors involved in stress tolerance in plants, such as W02005024028 and JP2006158402, which employ zinc finger transcription factors (Zinc finger) to confer water stress tolerance in plants. In turn, the document US2008163397 used the transcription factor CAAT-binding, conferring tolerance to drought and cold stress, using the B domain of the transcription factor. Likewise, in patent US2009265813 the transcription factor AP2 is used to obtain transgenic plants related to multiple characteristics, including enhanced root growth and obtaining drought tolerance as a final result.

The patents mentioned here reflect the biotechnological potential of 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 the perception of water stress, allowing the plant to anticipate and accelerate defense mechanisms. No document presents protection on genes related to the Scdr2 gene, whose sequence is described in SEQ ID: 1 (Annex 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 stresses (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 Cl, Daros E, de Campos MKF, de Carvalho JFRP, et al (2007) Evaluation of the stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress. Physiologia Plantarum 130: 218-229). And the use of sugarcane genes that give tolerance to abiotic stresses, as opposed to genes from other species, allows the production of transgenic sugarcane plants containing genes from the sugarcane itself, which have greater acceptance by consumers. In fact, 70% of consumers support genetic modifications involving genes from their own species, in contrast to 26% support when dealing with genes from other species (Rommens CM (2010) Barriers and paths to market for genetically engineered crops. Plant Biotechnology Journal 8:101-111).

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

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a method of producing plants that contain in their cells a sequence of sugarcane nucleotides and the expression of this gene leads to greater tolerance to abiotic stresses in the plant in question.

In a broader form, the polynucleotide encoding the sugarcane protein is expressed by a promoter and terminator that function in plants. More specifically, the present invention provides a recombinant DNA polynucleotide in the 5' to 3' direction, which comprises a promoter that functions in plants, operably linked to a second polynucleotide that encodes a sugarcane protein, operably linked to the terminator that terminates transcription. of the DNA polynucleotide providing a polyadenylation site.

The invention also describes a recombinant DNA sequence in which the promoter is selected from the group consisting of inducible promoters, constitutive promoters, temporally regulated promoters, tissue-preferred promoters, stress-specific promoters, drought-inducible promoters, deficit-inducible promoters. water, and tissue-specific promoters.

Also described are plant cells and plants that contain recombinant DNA molecules in their genome, as described, and the propagules and descendants produced from them. Plants include, but are not limited to, crop plants, monocots or dicots and may include sugarcane, soybeans, corn, canola, rice, cotton, barley, oats, grass, wheat, jatropha, mango, guava, lemon, avocado, orange, plum, pitta, jabuticaba, apple, peach, potato, pea, tomato, rose, sunflower, beans, eucalyptus, avocado, strawberry, pear, apple, guava, cocoa, lemon, passion fruit, forage palm, castor bean, manioc, rubber tree, mate, rosewood, coffee, pumpkin, watermelon, peach, pitanga and cashew.

The invention also provides a method for producing plants tolerant to abiotic stresses, thanks to genetic transformation with a recombinant DNA molecule that expresses a sugarcane protein, as well as the 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 saline stress would limit the growth of an untransformed plant of the same species, a higher growth rate under conditions where water would be limiting for the growth of an untransformed 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 for the growth of an untransformed plant of the same species. same species, higher percentage of plants surviving after a prolonged period of drought or under salt stress than an untransformed plant of the same species, a higher yield when compared to an untransformed plant of the same species, or greater drought tolerance in compared to an untransformed plant of the same species.

The present invention comprises the propagation of plants of this invention, for example, with the purpose of generating seeds, by simply planting such seeds in the soil, or from sprouting, as is the case of using stalks in sugarcane. , and which allows these plants to grow, for example, under stress conditions. More specifically, this invention provides a method for producing a plant which has as one of the characteristics, such as tolerance to abiotic stresses, an increase or increment in root mass yield. The invention comprises the steps of inserting into the genome of a plant cell, the construction of a recombinant DNA molecule comprising a sugarcane gene, the obtaining of a transformed plant cell or transformed cells, the regeneration of transformed plants from plant cells and the selection of plants that present better agronomic characteristics. One of the features of the invention is that the selected plants show greater 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: Evaluation of the expression by real-time PCR 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 V4: SP86-155. VI and V3 are tolerant sugarcane varieties, and V2 and V4 are sensitive sugarcane varieties. The asterisk indicates samples with values with a statistically significant difference.

Figure 2: Effect of mannitol and NaCl on germination of plants containing SEQ ID NO: 1. Percentage of germination of transgenic tobacco plants containing SEQ ID NO: 1 (Scdr2-1, Scdr2-2 and Scdr2-3) and wild plants (wt) at 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 3: Effects of salt 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 they were submitted to 10 days of irrigation with 200 mM mannitol or 175 mM NaCI and after that they were rehydrated for 3 days with water, a- c: photosynthesis (A), df: Internal concentration of CO2 (Ci); g-i: stomatal conductance (gs); j-l: transpiration 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 4: Water content in stressed leaves of wild plants and transgenic plants containing SEQ ID NO: 1. 30-day-old plants were exposed for 10 days to 200 mM mannitol or 175 mM NaCl, and after stress they were irrigated with water for 3 days. In the control treatment, the plants were irrigated throughout the experiment with water (n=5).

Figure 5: Effects of drought and salt stress on dry mass of wild-type plants and transgenic plants containing SEQ ID NO: 1. 30-day-old plants were exposed for 10 days to 200 mM mannitol or 175 mM NaCl, and rehydrated for 3 days with Water.

Figure 6: Phenotype of wild (wt) tobacco plants and transgenic plants containing SEQ ID NO: 1 maintained under water and salt stress. Top row: wt and three independent event plants containing SEQ ID NO: 1 (Scdr2-1, Scdr2-2 and Scdr2-3) grown under normal conditions for 5 weeks. Middle row: 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.

BRIEF DESCRIPTION OF ANNEXES

Annex 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.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a method for producing transgenic plants tolerant to environmental stresses thanks to the introduction of a gene that encodes a new sugarcane protein, whose function was previously unknown. The gene, named Scdr2 (drought-related sugarcane), is described in the sequence SEQ ID NO: 1 (Appendix 1A), and the protein deduced from this DNA sequence is shown in SEQ ID NO:2 (Appendix 1B).

The invention described in this document relates to a recombinant DNA vector comprising the nucleotide sequence SEQ ID NO: 1, useful for producing transgenic plants and a method for producing genetically modified plants that overexpress a nucleic acid molecule, a novel gene. from sugarcane, which comprises SEQ ID NO: 1, involved in the response of plants to abiotic stresses, and whose overexpression produces a significant improvement in the plant's 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 the production of plants that are tolerant of such abiotic stresses. Likewise, the invention comprises sequences with an identity equal to or greater than 60% to SEQ ID NO: 1, but not limited thereto.

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

The plant transformation vector used to introduce the nucleic acid into the plant cell may be a plasmid, in which DNA SEQ ID NO:1 is inserted into restriction endonuclease cleavage sites or by recombination mediated by other types of protein. The DNA is inserted into the cloning vector using easily 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 Education 11: 82-82). The resulting plasmid, which includes SEQ ID NO:1, can then be used to transform a plant cell, plants, or plant parts by conventional transformation methods.

For plant transformation, the plasmid preferably also includes a selectable marker gene in plant transformation, although there are methods that dispense with the use of this type of 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-enolpyruvylshikimate gene -3-phosphate synthase (EPSPS), or acetolactate synthase (ALS) gene. In the present invention, the selection marker is the Nptll gene, which allows the selection of transformants with kanamycin.

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

The plasmid preferably also includes promoters suitable for the expression of the nucleic acid indicated in SEQ ID NO: 1 and also for the expression of the selection marker gene as well as the reporter gene. The cauliflower mosaic virus 35S (CaMV 35S) promoter is commonly used for plant transformation, as is the rice actin 1 gene (Actl), the ubiquitin 1 gene (Ubil), 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 can also be used.

In the plasmid, the DNA described in SEQ ID NO:1 may be under the control of a constitutive promoter or it may be under the control of the promoter of the same gene or a different promoter. For plant transformation, the plasmid preferably also includes a terminator-encoding nucleic acid molecule, such as the 3' non-coding region of genes encoding an actin protease inhibitor, cauliflower mosaic virus, or cauliflower mosaic virus. nopaline (NOS). In the present invention, the plasmid includes the nopaline synthase (NOS) terminator.

The plasmid is preferably a binary plant transformation vector in which the genes of interest are inserted within the borders of the T-DNA. Examples of such plant transformation vectors that can be used in the present invention are vectors obtained from commercial sources, such as the pCambia, pBH21 or pGreenII series which contain a low replication RK2 origin, the neomycin phosphotransferase marker gene ( nptII), and the nopaline synthase (NOS) terminator, constitutive promoter, and a 3' NOS polyadenylation signal.

For plant transformation, any suitable method for transforming monocots or dicots can be used, for example Agrobacterium-mediated transformation or particle bombardment (also known as bioballistics transformation). In Agrobacterium-mediated transformation, plant cells are contacted with an inoculum of bacteria transformed with the plasmid containing the DNA indicated in SEQ ID NO: 1 of the invention, for example, by inoculating plant cells with a suspension of bacteria transformed. Bacteria of the genus Agrobacterium that can be used to transform plant cells include species of Agrobacterium rhizogenes, Agrobacterium tumefaciens, preferably strains of A. tumefaciensLBA4404, EHA105 or GV301. 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 in the binary vector are cultivated in a growth medium in the presence of antibiotics, such as, for example, kanamycin, for

select the bacterial cells that have the binary plasmid. Then, wild tobacco leaves are superficially sterilized, cut into small discs and incubated in a suspension of A. tumefaciens for a suitable time. Different fabrics can be used for the transformation, such as leaves, stem, flower, among others. After incubation with A. tumefaciens, the infected leaves are transferred to the in vitro culture medium for a certain period of time. The selection of transgenic plants is initiated by placing the infected plants in the in vitro antibiotic selection medium. After the development of seedlings with roots, they are transferred to soil and grown in a growth chamber.

Additionally, the present invention relates to the production of transgenic plants transformed with plasmids 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 to produce 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 that contain in their cells a chimeric gene capable of being expressed in plant cells, as well as subsequent generations comprising a DNA sequence of SEQ ID NO: 1 and DNA sequences that allow the expression of said protein in plant cells.

Transgenic plants according to the invention include, without limitation, cereals such as wheat, barley, maize, rice, oats, forage grasses, peat, other monocots such as sugar cane, miscanthus, or any other crop 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: ASSESSMENT OF SUGARCANE SCDR2 GENE EXPRESSION LEVEL

To assess the expression pattern of the Scdr2 gene under drought, a quantitative Real-time-PCR was performed as described by Rocha ef al (Rocha FR, Papini-Terzi FS, Nishiyama MY, Vencio RZN, Vicentini R, et al. (2007) Signal transduction-related responses to phytohormones and environmental challenges in sugarcane. Bmc Genomics 8) using leaves of sugarcane varieties with greater drought tolerance (SP83-5073 and SP83-2847) and two with greater drought sensitivity (SP86-155 and SP90- 1638) kept under irrigation or rainfed (with irrigation suspension, equivalent to drought stress). The sugarcane GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene was used as a reference gene.

Expression under dry (rainfed) was determined using the 2-ΔΔCT method (Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods 25: 402-408), using unstressed (irrigated) samples as a control. The statistical significance of the 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. The Expression profile was considered valid when P>0.95. For each treatment, leaves of six plants were used.

The result of the analysis showed that the expression of the Scdr2 gene was strongly induced after 24 hours of stress in drought tolerant varieties, and lately induced (72 hours) in sensitive varieties, which could explain the contrasting levels of drought tolerance (Figure 1). . These data suggested that the Scdr2 gene could be involved in drought tolerance in sugarcane.

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

A clone with the 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 (Appendix 1), was amplified by PCR using specific oligonucleotides 5'-GGATCCCTCATCGCCAGCTCCCAT-3-' and 5'-TCTAGACCTGTGCAGTGTCGGATTATTC-3', and cloned into the pGEMT vector -Easy (Promega, USA).

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

Example 3: PRODUCTION OF GENETICALLY MODIFIED PLANTS

Wild tobacco seeds (Nicotiana tabacum, var. SR1) were germinated in Petri dishes containing Murashige-Skoog (MS) medium with 0.9% (w/v) agar. Seedlings were transplanted into soil composed of Bacto (Michigan 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 into 1 L pots containing the same mixture mentioned above.

The plants were grown in a growth chamber with a photoperiod of 16/8h light/dark (300-400 μmol photons m'2s-1) at 25°C and relative humidity of 75-80%. For seed production, plants were induced to flower by extending the time of exposure to light.

Wild tobacco leaves were surface sterilized, cut into small discs and incubated in a suspension of A. tumefaciens for 5 to 20 min. After that, the infected leaves were transferred to MS medium (supplemented with 2 mg L'1 benzyl-adenine and 0.1 mg L'1 naphthylacetic acid) for 3 days.

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

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

The initial characterization of plants rooted and transferred to soil was carried out primarily by the histochemical assay using X-Gluc as a substrate for the detection of the beta-glucuronidase gene, also inserted in the expression cassette.

It was observed that a large number of plants showed strong blue staining, 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 as a template.

Then, total DNA was extracted from leaf samples of wild plants and transgenic plants. For this, about 50 - 150 mg of leaves were collected, which were quickly transferred to a mortar containing liquid nitrogen. After evaporation in nitrogen, the material was pulverized until obtaining a very fine powder, to which 3 mL of extraction buffer was added. Immediately the solution was transferred to 1.5 mL sterile plastic tubes, which were kept in a water bath at 65°C for 30 minutes, with gentle agitation every 10 minutes.

Soon after, 10 microliters of Proteinase K were added to the tubes and they were incubated again in a water bath at 65°C for 30 minutes, with gentle agitation every 10 minutes. Then, the tubes were 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 phenol-chloroform (1:1), at a ratio of 1:1 (1 part supernatant: 1 part phenol-chloroform).

In sequence, the tube was gently inverted several times to obtain 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. Soon after, 100 mg/mL RNAse (1:100) was added and the mixture was incubated at 37°C for 30 minutes.

In the last phase of the extraction, the same volume of chlorophyll solution (24 parts of chloroform: 1 part of isoamyl alcohol) was added and the mixture was placed under gentle agitation 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 the chlorophyll solution was added and the mixture was gently stirred. 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, 1 volume of isopropyl alcohol was added and the solution was then homogenized until a DNA precipitate formed. The precipitated DNA was washed in the tube itself in 70% alcohol, 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 tobacco plants was confirmed by polymerase chain reaction (PCR). Initially, lμL of DNA, 1.5μL 4 dNTP (1OmM), 1.4μL MgCl2 (25mM), lμL of Sense Primer (10μM), lμL Antisense Primer (10μM), 2.5μL 1Ox Taq Buffer, 0.3μL Taq were used Polymerase (Fermentas), 16.3μL of MilliQ 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 a 0.8% agarose gel in TAE containing ethidium bromide, under ultraviolet light. Among 12 samples of supposedly transgenic plants, the presence of SEQ ID NO:1 was confirmed in 8. These plants were used in further assays to observe the effects of the Scdr2 gene indicated in SEQ ID NO:1 on the characteristics of tobacco plants.

Example 5: ANALYSIS OF SEED GERMINATION OF GENETICALLY MODIFIED PLANTS CONTAINING SEQ ID NO: 1 UNDER WATER STRESS CONDITIONS

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

The seeds were superficially sterilized with 70% alcohol for 1 min, incubated in 2% NaClO for 30 min and washed five to six times in sterile distilled water. The seeds were sown in Petri dishes (30 seeds per dish) containing solid Murashige-Skoog (MS) medium, pH 5.8, in a chamber at 23°C with photoperiod 16 / 8 h light/dark (300-400 mmol photons m'2 s1).

Then, the seeds that were transformed in three independent events (Scdr2-1, Scdr2-2 and Scdr2-3), were used to evaluate the percentage of germination in culture medium containing mannitol, verifying the response to water 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 emergence of the hypocotyl.

In the control condition without mannitol, seeds from wild plants and plants containing SEQ ID NO: 1 showed similar germination percentage (Figure 2A). Water stress inhibits or reduces germination in tobacco plants. In the medium containing 200 mM mannitol seeds from plants containing SEQ ID NO: 1 showed faster germination compared to wild-type seeds (Figure 2B). Under conditions of higher water stress (300 mM), germination of wild plants, however, transgenic plants reached approximately 60% (Figure 2C). Therefore, plants with the Scdr2 gene indicated in SEQ ID NO: 1 showed a higher level of tolerance to water stress.

In numerous cultivated plants, germination and the initial growth of seedlings 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 medium significantly delay germination and reduce the rate of germination events. The low rate of seed germination can result in irregular establishment in the soil, and reduced productivity. A desirable plant characteristic for the production of commercial cultivars under field conditions is the ability of the seed to germinate quickly and homogeneously 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 GERMINATION UNDER SALINE STRESS

To determine the effects of saline water stress on germination, seeds from transgenic 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% NaClO for 30 min and washed five to six times in sterile distilled water. The seeds were sown in Petri dishes (30 seeds per dish) containing solid Murashige-Skoog (MS) medium, pH 5.8, in a chamber at 23°C with a photoperiod of 16/8 h light/dark (300-400 mmol photons m'2 s'1) and to test the effect of salt stress, NaCI (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 appearance of the hypocotyl.

When seeds were plated in 100 mM NaCl the transgenic plants containing SEQ ID NO: 1 germinated much faster than wild-type plants. The 50% germination of the transgenic plants was reached on the seventh day, while the wild plants reached 50% only on the eleventh day 11 (Figure 2D). However, using a more severe stress concentration (175 mM NaCI), germination was totally 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 saline stress.

Different responses to abiotic stresses are the result of cooperative interactions between the various biochemical, physiological and morphological character components. These interactions can vary between species and even varieties, as observed 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 phases that are very sensitive to stressful conditions. In turn, soil salinity is a global problem, which not only prevents the use of large areas for agriculture, but can also reduce productivity in certain agricultural areas, as a result of the increase in salinity due to irrigation with inappropriate water by have a high salt content.

Under high salinity conditions, while wild plants showed total inhibition of germination, transgenic plants containing SEQ ID NO: 1 showed a higher percentage of germination. Consequently, this invention shows that SEQ ID NO: 1 is useful for producing genetically modified plants with increased 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 pots containing Plantmax HT (Eucatex, Brazil) in a growth chamber at 22°C and 18 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 per 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 the 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 C02 (Ci), under control conditions and under water stress, fully expanded leaves were used in the same position of the plants of tobacco.

Three independent events were evaluated from transgenic plants containing SEQ ID NO:_1 and wild-type plants. Measurements were performed with IRGA equipment (LCpro+; ADC bioscientific, United Kingdom), at a CO2 concentration of 360 μL L'1, light intensity of 1000 μmol m^s'1, gas flow of 200 mL min1 and temperature inside the chamber. 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. Journal of Plant Physiology 165:1745-1755 ).

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

Likewise, both gs (Figure 3H) and E (Figure 3K) were rapidly recovered during rehydration after water stress. Ci showed similar values under normal conditions, while under stress conditions plants containing SEQ ID NO: 1 performed better than wild plants (Figure 3E). In summary, a clear difference between transgenic and wild plants was observed in all photosynthetic parameters evaluated under drought conditions.

Agricultural crops usually experience water scarcity at some point throughout their life cycle and, depending on the duration and intensity of water stress, significant losses in productivity can 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 in conditions of water scarcity.

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

To evaluate the effect of SEQ ID NO: 1 under salt stress conditions, 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 and 18 hours of light. The plants were irrigated daily with 100 ml of water for 4 weeks before stress began.

To assess the effect of salt stress, transgenic and wild-type plants were regularly irrigated with water for five weeks (control treatment), while a group of plants was irrigated with 175 mM NaCI for 10 days and then rehydrated for three days with water (in the control plants were kept under irrigation during the time of the experiment).

Three independent events from 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 control conditions and under saline stress.

The assessment 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 show such recovery ( Figure 3C). In turn, both gs (Figure 31) and E (Figure 3L) were rapidly recovered during rehydration after saline stress. Ci showed similar values under normal conditions, although under stress conditions plants containing SEQ ID NO: 1 performed better than wild plants (Figure 3F).

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 RELATIVE WATER CONTENT OF TRANSGENIC PLANTS CONTAINING SEQ ID NO: 1 SUBJECTED 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.

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 the fresh mass and DW the dry weight. Under water stress the water content in leaves was drastically reduced in wild plants (Figure 4). In contrast, transgenic plants containing SEQ ID NO: 1 were able to maintain their water content almost unchanged, indicating that SEQ ID NO: 1 confers a greater ability to maintain turgor under water stress conditions.

Example 10: ASSESSMENT OF THE RELATIVE WATER CONTENT OF TRANSGENIC PLANTS CONTAINING SEQ ID NO: 1 SUBJECTED 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, being 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 assess their dry weight. The water content in the leaf was calculated as 100x(FW-DW) /(FW), with FW being the fresh mass and DW the dry weight.

Salt stress decreased water content in wild plants, while transgenic plants containing SEQ ID NO: 1 were able to maintain almost unchanged leaf water content (Figure 4), indicating that plants that overexpress Scdr2 are able to to maintain turgor under stress conditions.

Example 11: EVALUATION OF THE EFFECT OF SEQ ID NO: 1 ON THE DRY MASS OF PLANTS SUBJECTED TO WATER STRESS.

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 placed 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 they were 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 the salt, drought, oxidation and cold tolerance. Plant Molecular Biology 68:131-143).

Plant samples were collected and kept at 80 °C for 24 hours to evaluate the dry mass in the roots and shoots, 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 5).

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

Wild tobacco plants and transgenic plants containing SEQ ID NO: 1 were germinated for 16 days and then transferred to pots containing Plantmax HT (Eucatex, Brazil), remained 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 starts. For saline stress, the plants were irrigated with 100 ml of 175 mM NaCI for 10 days, and then they were 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 the salt, drought, oxidation and cold tolerance. Plant Molecular Biology 68:131-143).

Samples of the transformed plants were collected and kept at 80 °C for 24 hours to evaluate the dry mass in the roots and shoots, 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 ).

It was observed that saline stress reduces the dry mass of wild and transgenic plants. However, the presence of SEQ ID NO: 1 in the transgenic plants was associated with a greater ability to accumulate dry matter under saline stress conditions (Figure 5).

Example 13: VISUAL INSPECTION OF THE EFFECT OF WATER AND SALINE STRESS ON WILD TOBACCO PLANTS AND TRANSGENIC PLANTS CONTAINING SEQ ID NO: 1.

To evaluate the response to water and salt stress, 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 starts. To assess the effect of water stress, seedlings were irrigated with 100 ml of 200 mM mannitol for 10 days and then again irrigated 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 the salt, drought, oxidation and 5 cold tolerance. Plant Molecular Biology 68: 131-143) and to evaluate the effect of salt stress the plants were irrigated with 175 mM NaCI for 10 days and later they were rehydrated for three days with water. In the control treatment, the plants were irrigated during the time of the experiment.

Under control conditions, all plants had similar results (figure 6), 10 while under water and saline stress the leaves of wild plants were wilted. In turn, transgenic plants containing SEQ ID NO: 1 exhibited a phenotype practically similar to plants maintained without stress (figure 6).

These same plants were used in the analyzes shown in examples 7 and 8 and are in agreement with the best performance of the plants in terms of photosynthetic parameters. Likewise, this phenotype seen in Figure 6 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 (16)

1. Method for producing genetically modified plants, characterized in that it comprises the following single step: a) Production of a vector comprising the sequence described in SEQ ID NO:1, operably linked to a plant functional promoter and a functional terminator in plants inserted into the genome of a plant cell, plant or plant parts. 2. Method, according to claim 1, characterized in that the genetically modified plants present in their genome the gene sequence SEQ ID NO:1. 3. Method according to claim 1, characterized in that the gene sequence SEQ ID NO:1 is obtained from sugarcane. Method according to claims 1 and 2, characterized in that it can be applied to monocots. 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, soy, corn, rice and wheat. Method, according to claims 1 and 2, characterized in that it allows the vector to be integrated into the genome of the plant cell. Method, according to claims 1 and 2, characterized by modulating the plant's tolerance to environmental stresses. 10. Method according to claim 11, characterized in that the environmental stress is drought. 11. Method according to claim 11, characterized in that the environmental stress is salinity. 12. Recombinant DNA vector, characterized by comprising all the following elements: a. polynucleotide sequence defined by SEQ ID NO:1, which encodes the polypeptide defined by SEQ ID NO:2; B. Scdr2 gene defined by SEQ ID NO:1 inserted between the edges of the T-DNA of a binary vector; ç. heterologous promoter operably linked to the gene sequence SEQ ID NO:1 ser CaMV 35 S from cauliflower mosaic virus; d. terminator operatively linked to the gene sequence SEQ ID NO:1 and the nucleic acid encoding the terminator being that of nopaline synthase (NOS); and. NptII marker gene; f. reporter gene; g. DNA transfer for creating transgenic plant cell, plant or plant parts. 13. Recombinant DNA vector, according to claim 12, characterized in that it is the expression or cloning or plasmid vector. 14. Use of the method described in claims 1 to 11, characterized in that it is applied for the production of plant cell, plant or plant parts. 15. Use of the vector described in claim 12 characterized by producing transgenic plants tolerant to environmental water stress. 16. Use of the gene sequence described in SEQ ID NO: 1 characterized by encoding the polypeptide defined by SEQ ID NO:2 and being applied in monocots or dicots.

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