Senescence-induced promoter, method of producing transgenic plants containing the promoter, expression vector and use.

Abstract

The present invention relates to the field of molecular biology and transgenics. More specifically, the present invention relates to the control and regulation of gene expression in plant cells using the senescence-induced p1 promoter, which allows the expression of genes capable of expressing proteins involved in plant breeding, preferably sugarcane. The invention also deals with the method of producing transgenic plants through the insertion of a gene expression vector comprising said promoter associated with genes of interest; also comprising the gene expression vector, transgenic plants modified by them and the use of the vector comprising the p1 promoter in combination with other genes.

Description

1 / 13 Senescence-induced promoter, method of producing transgenic plants containing the promoter, expression vector and use. Field of Invention

[001] The present invention relates to the field of Molecular Biology and transgenics. More specifically, the present invention relates to the control and regulation of gene expression in plant cells using a PI promoter induced by natural senescence, which allows the expression of genes capable of expressing proteins involved in plant breeding, preferably sugarcane. The invention also deals with the method of producing transgenic plants through the insertion of a gene expression vector comprising said promoter associated with genes of interest; also comprising the gene expression vector, transgenic plants modified by them and the use of the vector comprising the PI promoter in combination with other genes. Fundamentals of the Invention

[002] Senescence is the age-dependent deterioration of plant cells, leading to cell death and the end of the plant's life cycle. In addition to the natural senescence process, biotic and abiotic factors are known to activate senescence pathways. Leaf senescence constitutes the final stage of development of Petition 870180153003, dated 11 / 20 / 2018, page 7 / 31 2 / 13 of the leaf, and the earliest and most important event during this process is chloroplast degradation. Consequently, there is a decrease in the photosynthetic rate and chlorophyll content, with subsequent yellowing of the leaves. During senescence, some compounds released from degraded chloroplasts, mainly nitrogen, and compounds released from other organelles are remobilized to other developing tissues, such as young leaves or fruits and grains. The optimal use of nutrients accumulated during the photosynthetic period is fundamental for the plant's adaptation, which is critically affected by the fine control of the leaf senescence process to ensure its effective remobilization. In several higher plant species, leaf senescence progresses from the tip towards the base of the leaf blade, indicating the direction of nutrient flow.Senescence is a tightly controlled process, and therefore many genes associated with senescence are finely regulated during this process.

[003] The study of leaf senescence in different crops contributes not only to the understanding of this important natural process, but also to the development of new strategies for its control, improving the agricultural characteristics of these crops. For example, studying the dynamics of cell wall synthesis and degradation during sugarcane senescence can help in the development of biotechnological tools to aid in the production of bioethanol from lignocellulosic biomass. Furthermore, plant senescence pathways can be activated by biotic and abiotic stresses, and understanding them under these conditions... Petition 870180153003, dated 11 / 20 / 2018, page 8 / 31 3 / 13 natural offers a suitable strategy for generating transgenic crops capable of dealing with these stresses.

[004] Thus, the present invention is based on a promoter that can be associated with genes involved in cytokinin biosynthesis or in increasing productivity to drive the expression of these genes in genetically modified plants, preferably sugarcane. Description of the State of the Art

[005] The process of obtaining plants through genetic improvement has been increasingly explored due to the challenges related to increasing production and productivity, controlling pests and diseases, and improving product quality. Therefore, currently, several patent documents can be found that describe technologies aimed at this purpose. As an example, we can cite document KR101845248, which refers to a transgenic plant in which a MYC2 gene operatively linked to a promoter is introduced to inhibit senescence; a method for producing the transgenic plant; a composition for inhibiting plant senescence comprising the MYC2 gene operatively linked to the promoter; a kit for inhibiting plant senescence comprising the composition; and a method for inhibiting plant senescence comprising a step to introduce the composition into the plant that will express it.When the method of the present invention for inhibiting plant senescence is used, the level of chlorophyll contained in the plant leaves is maintained and improved. Petition 870180153003, dated 11 / 20 / 2018, p. 9 / 31 4 / 13 photosynthetic efficiency. Document US20150176021 provides methods for increasing nitrogen use efficiency, fertilizer use efficiency, yield, growth rate, vigor, biomass, oil content and / or tolerance to abiotic stress. Isolated polynucleotides and polypeptides can be used to increase nitrogen use efficiency, fertilizer use efficiency, yield, growth rate, vigor, biomass, oil content and / or tolerance to abiotic stress in plants. Document WO2014036048 describes methods and compositions useful in altering gene expression. The invention provides polynucleotides useful for altering gene expression, as well as cells, plants and seeds comprising the polynucleotides. The invention also provides methods for using lincRNAs to alter gene expression. US patent document US2007292874 refers to the identification of 348 genes associated with sucrose accumulation in sugarcane plants. Differentially expressed genes identified in plants with high and low sucrose concentrations were obtained using cDNA microarray and quantitative PCR technologies. The prospected genes can be used to identify, distinguish, characterize, and / or develop plants with higher sucrose content. KR patent document KR20180038717 describes the MtATPG1 protein, which induces increased yield and delayed senescence in plants, the gene that expresses this protein, MtATPG1, and its utilization. The document demonstrates that plants in which the gene is introduced and expressed improve productivity and delay senescence. Petition 870180153003, dated 11 / 20 / 2018, p. 10 / 31 5 / 13

[006] However, prior art documents do not compromise the technology presented here, which deals with a P1 promoter induced by natural senescence, which can be introduced into plants to drive the expression of genes of interest, preferably the ipt gene. The present invention also deals with the expression vector comprising the promoter and the genes of interest, as well as its use, transgenic plants containing this vector and the method of obtaining these plants. The promoter developed here can be used in combination with different genes, depending on the specific objectives of each goal. Its combination with the ipt gene, for example, leads to a delay in the senescence process and a consequent increase in biomass production, flowers, seeds, fruits, shelf life, and tolerance to abiotic stresses. Its combination with cell wall genes increases biomass digestibility. The use of the technology presented here allows for the production of crops with structural and utilization advantages. In the case of sugarcane production for the sugar-energy sector, for example, the use of the P1 promoter associated with the ipt gene could improve ethanol production per unit area. The idea is to increase the release of sugars from the biomass. Since sugarcane is harvested for alcohol production when the plant is older, the cell wall is more rigid, and the plant produces more biomass.One of the alternatives sought with this technology is to express important enzymes under the control of promoters induced by natural senescence. In this way, from the moment the plant ages, these... Petition 870180153003, dated 11 / 20 / 2018, page 11 / 31 6 / 13 enzymes are produced, making the plant less recalcitrant and improving alcohol production.

[007] Therefore, the P1 promoter, the vector containing this promoter, and the method presented here can be used to provide improvements to crops, preferably sugarcane crops, with these improvements being adapted to the needs and particularities of each one. Brief description of the figures

[008] Figure 1: Binary vector containing the P1 expression cassette (SAG12-like promoter from sugarcane) + Gus reporter gene.

[009] Figure 2: Histochemical assay of Setaria viridis transformed with vector P1 by means of Agrobacterium tumefaciens. A, E and I untransformed control plant. B, F and J plant transformed in phase 3. C, G and L plant transformed in phase 2. D, H and M plant transformed in phase 1. A, B, C and D - root, E, F, G and H - leaf +1, I, J, L and M - leaf +8. Detailed description of the invention

[0010] The present invention relates to a gene promoter (SEQ ID No. 1), whose activity is induced by natural senescence, in order to allow the expression of genes, Petition 870180153003, dated 11 / 20 / 2018, page 12 / 31 7 / 13 preferably the ipt gene (SEQ ID No. 6), capable of expressing proteins involved in plant breeding, preferably sugarcane. The invention also deals with a method for producing transgenic plants by inserting into these plants an expression vector comprising the P1 promoter represented by the sequence described in SEQ ID No. 1 or by sequences with 70 to 100% similarity to SEQ ID No. 1, associated with genes of interest; it also protects the expression vector containing this promoter associated with genes of interest, transgenic plants modified by them, and the use of the vector comprising the P1 promoter in combination with other genes. Furthermore, the technology protects an expression cassette comprising the gene promoter represented by the SEQ ID No. 1 sequence, its similar sequences, and genes of interest linked to these sequences.

[0011] The method for producing transgenic plants involves the following steps: insertion into cells of a promoter with at least 70 to 100% identity to the P1 promoter described in SEQ ID No. 1, which promotes the expression of genes of interest, preferably the ipt gene (SEQ ID No. 6); growth and regeneration of plant cells in specific media; selection of transformed plants.

[0012] Promoter P1 can be replaced by sequences with at least 70% identity to the sequence described in SEQ ID No. 1.

[0013] The transgenic plants produced are primarily sugarcane.

[0014] The gene expression vector comprises in its sequence a promoter with 70 to 100% identity to the promoter described in SEQ ID No. 1, associated with genes of Petition 870180153003, dated 11 / 20 / 2018, page 13 / 31 8 / 13 of interest, preferably the ipt gene, as well as transgenic plants modified by these gene expression vectors and the use of these vectors. The genes involved in senescence can be selected from the group comprising the ipt gene and cell wall genes represented by the sequences described in SEQ ID No. 6 to SEQ ID No. 12, and by sequences that show 70 to 99% similarity to these genes.

[0015] Here are some concepts to help you better understand the technology.

[0016] A gene refers to the nucleotide fragment that expresses a specific protein, including regulatory sequences preceding (5' untranslated region) and following (3' untranslated region) the coding region. A native gene refers to an isolated gene with its own regulatory sequence found in nature. A chimeric gene refers to a gene comprising heterogeneous coding, regulatory, and regulatory sequences not found in nature. An endogenous gene refers to a native gene normally found in its natural location in the genome and is not isolated. An exogenous gene refers to a gene that is not normally found in the host organism but is introduced by gene transfer. A pseudogene refers to a nucleotide sequence that does not code for a functional enzyme.

[0017] Promoter refers to the DNA sequence in a gene, usually located upstream of the coding sequence, which controls the expression of the coding sequence by promoting recognition by RNA polymerase and other factors required for transcription itself. In an artificial DNA construct, Petition 870180153003, dated 11 / 20 / 2018, page 14 / 31 9 / 13 Promoters can also be used to transcribe dsRNA. Promoters may also contain DNA sequences that are involved in binding protein factors which control the effect of transcription initiation in response to physiological or developmental conditions.

[0018] Plants refer to photosynthetic organisms, both eukaryotic and prokaryotic, where the term developed plants refers to eukaryotic plants. The nucleic acids of the invention can be used to confer desired traits in essentially any plant. Therefore, the invention has use on various species of plants, including species of the genera Anacardium, Anona, Arachis, Artocarpus, Asparagus, Atropa, Avena, Brassica, Carica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoseyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panieum, Pannesetum, Passiflora, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Psidium, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.

[0019] The transcription termination signal and polyadenylation region of the present invention includes, but is not limited to, SV40 termination signal, HSV TK adenylation signal, Agrobacterium tumefaciens nopalin synthase gene termination signal (nos), CaMV 35S RNA gene termination signal, Trifolium subterranean virus termination signal Petition 870180153003, dated 11 / 20 / 2018, page 15 / 31 10 / 13 (SCSV), termination signal of the trpC gene of Aspergillus nidulans, and others similar.

[0020] “Gene expression vectors” are cloning vectors that possess all the genetic elements that allow the expression of recombinant proteins. EXAMPLES

[0021] The invention may be better understood through the following examples, which are not limiting. Example 1 - Isolation of the promoter

[0022] The promoter (herein referred to as P1 and represented by the SEQ ID No. 1 sequence) was isolated using the 5'race technique, combined with a simplified nested PCR technique using the Genome Walker Universal Kit (Clontech) according to the manufacturer's specifications. The following primers were used for amplification: 1. Reaction: Primer forward (SEQ ID NO. 2): 5'-GTAATACGACTCACTATAGGGC—3' Reverse primer (SEQ ID NO. 3): 5'-GTTCAGGGAAAACCTCCGCTACATC3' 2areação: Primer forward (SEQ ID NO. 4): 5'-ACTATAGGGCACGCGTGGT—3' Reverse primer (SEQ ID NO. 5): 5'-GTCGATCATCTCCTACGGGGAG-3' Petition 870180153003, dated 11 / 20 / 2018, pp. 16 / 31 11 / 13

[0023] The fragments obtained were cloned and sequenced using the dideoxynucleotide method (Sanger et al., 1979).

[0024] Example 2 - Cloning in binary vectors

[0025] Cloning into suitable binary vectors for monocotyledon transformation was performed by the company DNA Cloning Service, which synthesized the vector shown in Figure 1. This vector contains the P1 promoter, represented by SEQ ID No. 1, downstream of the coding sequence of the gus reporter gene.

[0026] Example 3 - Transformation into sectarian

[0027] The transformation of Setaria was carried out according to the protocol described by Martins et al, 2015 (Martins, PK, Ribeiro, AP, Cunha, BADB, Kobayashi, AK, Molinari, HBC A simple and highly efficient Agrobacterium-mediated transformation protocol for Setaria viridis. Biotechnology Reports 6 (2015) 41-44). In summary, seeds of Setaria viridis (accession A10.1) were planted in soil and cultivated in growth chambers with a relative humidity of 65% and a light intensity of 400 gmol m-2s-1, for 16 hours a day for approximately two months until the plants produced new seeds. These seeds were used for embryogenic callus induction after the removal of straw and sludge, disinfection with a 10% (v / v) sodium hypochlorite solution followed by a 0.1% Tween 20 solution and five washes in sterile distilled water. After this procedure, the dehulled seeds were placed in callus induction medium (CIM) and after 3-5 weeks in the dark at 25°C, the calluses... Petition 870180153003, dated 11 / 20 / 2018, page 17 / 31 12 / 13 were divided into small explants and subcultured in fresh CIM. After 4-5 days, these explants were used for transformation with Agrobacterium tumefaciens. For this, the calluses were co-cultured with Agrobacterium suspensions containing the vector mentioned in Figure 1 for three days in the dark at 22°C. After this step, the plant material was placed in selective medium, and one week later the explants were placed in selective regeneration medium and maintained at 25°C, 16h photoperiod, and light intensity of 100 pmol m⁻² s⁻¹. The resistant plantlets were then transferred to magenta plants until they reached 50 mm in height and then transplanted into pots with soil for acclimatization. These plants constituted the T0 generation and were tested for transgene stability up to the T2 generation, the stage at which the progeny is homozygous. The T2 plants were then analyzed for the expression of the Gus gene fused to the P1 promoter (SEQ ID No. 1).

[0028] Example 4 - Histochemical assays

[0029] GUS activity was verified according to classic histochemical assay protocols (Jefferson, RA Assaying chimeric genes in plants: the GUS gene fusion system, Plant Mol. Biol. 5 (1987) 387-405). Homozygous plants at various developmental stages transformed with the binary vector described in Figure 7 were subjected to tests of gus reporter gene activity. Plants at three developmental stages were tested: phase 1 (21 days), phase 2 (35 days), and phase 3 (42 days). Only plants in phase 3 showed strong gus activity, confirming the efficiency and specificity of the P1 promoter (SEQ ID No. 1) (Figure 2). Petition 870180153003, dated 11 / 20 / 2018, page 18 / 31 13 / 13

[0030] Example 5 - Analysis of Setaria viridis with the SAG12like::IPT construct

[0031] Setaria viridis plants in the T2 generation were analyzed for biomass accumulation and productivity. To induce the expression of the SAG12 promoter, plants in the third developmental phase (21 days after germination) were subjected to and maintained for 15 days in moderate drought (~50% available water in the soil). During the drought period, transgenic and non-transgenic plants were analyzed for photosynthetic pigment degradation, gas exchange, and gene expression analysis of genes involved in the senescence process (initial and final stages of senescence), cytokine, auxin, and ethylene levels. Transgenic plants containing the SAG12like::IPT construct (SEQ ID NO 12) showed less photosynthetic pigment degradation with a consequent increase in productivity. Petition 870180153003, dated 11 / 20 / 2018, page 19 / 31

Claims

1 / 2 CLAIMS 1- Expression cassette characterized by comprising the gene promoter described in SEQ ID No. 1 operationally linked to at least one gene of interest. 2- Expression cassette, according to claim 1, characterized in that the gene of interest has a sequence in accordance with the sequence described in any of the sequences described in SEQ ID No. 6 to SEQ ID No.

12. 3- Use of the gene expression cassette as described in claims 1 and 2, characterized by being in the production of transgenic plants, preferably sugarcane. 4- Method for obtaining transgenic plants characterized by comprising the following steps: a) Inserting, into plant cells, gene expression vectors containing the gene promoter sequence described in SEQ ID No. 1, operationally linked to at least one gene of interest; b) Growing or regenerating the plant cells in specific media; c) Selecting the transformed plants. 5- Method for obtaining transgenic plants, according to claim 4, characterized in that the gene of interest of the step consists of any of the sequences described in SEQ ID No. 8 to SEQ ID No.

12. Petition 870260008631, dated 29 / 01 / 2026, page 7 / 12 2 / 2 6- Method for obtaining transgenic plants, according to any one of claims 4 to 5, characterized in that the plant cells are preferably from sugarcane. 7- Gene expression vector characterized by comprising a gene promoter whose sequence is the sequence described in SEQ ID No.

1. 8- Gene expression vector, according to claim 7, characterized in that the promoter is associated with at least one gene of interest. 9- Gene expression vector, according to claim 8, characterized in that the genes of interest are selected from the group comprising the sequences described by SEQ ID No. 6 to SEQ ID No.

12. 10- Use of the gene expression vector as described in claims 7 to 9, characterized by its use in the production of transgenic plants that exhibit increased biomass and resistance to abiotic stresses, preferably sugarcane. Petition 870260008631, dated 01 / 29 / 2026, page 8 / 12

Images