Application of Setaria italica SiPYL3 gene and encoded protein thereof in regulation and control of plant stress resistance and / or yield

Abstract

The invention discloses an application of a millet SiPYL3 gene and an encoding protein thereof in regulation and control of plant stress resistance and / or yield. The invention relates to the field of plant genetic engineering, and provides application of SiPYL3 protein or a coding gene thereof in regulation and control of plant salt resistance and / or drought resistance and / or yield. The amino acid sequence of the SiPYL3 protein is as shown in SEQ ID No. 1. According to the invention, the coding gene of the SiPYL3 protein from millet is introduced into rice, so that the salt tolerance of the obtained transgenic line is improved, the drought resistance is reduced, and the yield is increased. Therefore, the SiPYL3 gene can positively regulate the salt tolerance and yield traits of the plant and negatively regulate the drought resistance of the plant. The invention has important significance for cultivating salt-resistant and high-yield plant varieties.

Description

Technical Field

[0001] The present invention relates to the field of plant genetic engineering, and particularly to the application of foxtail millet SiPYL3 genes and their encoded proteins in regulating plant stress resistance and / or yield. Background Art

[0002] Abscisic acid (ABA) is one of the main hormones in plants and plays an important role in regulating plant growth and development and responses to abiotic stresses (such as drought, low temperature, salt, etc.). As a receptor for ABA signals, the function of PYL proteins in Arabidopsis thaliana has been well elucidated. PYL is a soluble protein that recognizes ABA and then interacts with PP2C to form a PYL-ABA-PP2C complex. After the formation of this complex, the phosphorylation activity of the PP2C protein is inhibited, thereby releasing SnRK2, a protein kinase whose activity is inhibited by the interaction of PP2C with PP2C. Finally, the released SnRK2 is autophosphorylated and activated, phosphorylating transcription factors or downstream effectors to further conduct PYL-mediated ABA signal transduction.

[0003] Since PYL genes are a core component of ABA signals, they have been identified in many plants. For example, a total of 14 PYL genes have been identified in Arabidopsis thaliana, 46 in Brassica oleracea, 6 in sweet orange, 8 in grape, 12 in rice, and 27 have been identified in cotton. Among these PYL genes, some genes also play important roles in improving plant stress resistance and crop yield. For example, AtPYL4 and AtPYL9 overexpression of AtPYL8 and AtPYL9 in Arabidopsis thaliana seedlings improved drought resistance. In addition, OsPYL3 overexpression of OsPYL5 in Arabidopsis thaliana enhanced drought resistance and cold tolerance of seedlings. OsPYL8 and OsPYL9 play a positive regulatory role in seed germination, seedling growth, drought tolerance, and salt tolerance. In addition, ZmPYL9 and ZmPYL12 positively regulate the sensitivity of rice to ABA treatment during the germination period. Overexpression of PYL genes in multiple plants has been elucidated, but their functions in foxtail millet are still unclear.

[0004] Foxtail millet is one of the oldest crops in the world, with typical drought resistance and barren tolerance. In addition, foxtail millet has a small diploid genome (about 515 Mb; 2N = 18), and has the advantages of a short life cycle, self-pollination, and high seed production. The completion of genome sequencing has greatly promoted the research on the functional genomics of foxtail millet, making foxtail millet an ideal model crop for studying the mechanism of plant stress resistance. Mining the PYL related genes and analyzing their stress resistance functions can lay a foundation for further understanding the stress resistance regulation network of foxtail millet, and also provide new genetic resources for improving plant stress resistance. SUMMARY OF THE INVENTION

[0005] The object of the present invention is to provide the application of foxtail millet SiPYL3 genes and their encoded proteins in regulating plant stress resistance and / or yield.

[0006] In the first aspect, the present invention claims the application of the SiPYL3 protein or its encoding gene in all or part of the following P1-P3: P1, regulating plant salt tolerance; P2, regulating plant drought resistance; P3, regulating plant yield.

[0007] Wherein, the SiPYL3 protein can be any of the following: (A1) a protein with the amino acid sequence of SEQ ID No.1; (A2) a protein derived from foxtail millet that has the same function after substitution and / or deletion and / or addition of one or several amino acid residues in the amino acid sequence shown in SEQ ID No.1; (A3) a protein derived from foxtail millet that has the same function and has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the amino acid sequence defined in any of (A1)-(A2); (A4) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in any of (A1)-(A3).

[0008] In the above-mentioned protein, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using identity search sites on the Internet, such as the BLAST web page on the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0009] The above-mentioned identity of more than 80% can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The above-mentioned identity of more than 85% can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The above-mentioned identity of more than 90% can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The above-mentioned identity of more than 95% can be at least 95%, 96%, 97%, 98% or 99% identity.

[0010] Furthermore, in the above-mentioned application, increasing the expression level of the SiPYL3 protein in the plant (or SiPYL3 increasing gene expression), the salt tolerance of the plant is enhanced.

[0011] Furthermore, in the above-mentioned application, increasing the expression level of the SiPYL3 protein in the plant (or SiPYL3 increasing gene expression), the drought resistance of the plant is reduced.

[0012] Furthermore, in the above-mentioned application, increasing the expression level of the SiPYL3 protein in the plant (or SiPYL3 increasing gene expression), the yield of the plant is increased.

[0013] Among them, the plant may originally have no expression of the SiPYL3 protein, or may originally have the expression of the SiPYL3 protein, that is, the increase in the expression level of the SiPYL3 protein can be from none to some, or can be an increase on the original (non-zero) basis. The same applies hereinafter.

[0014] In a second aspect, the present invention claims the use of increasing the expression level of the SiPYL3 protein in a plant in all or part of the following p1 - p3: p1, regulating the root length and / or shoot length of a plant under salt stress; p2, regulating the survival rate of a plant under drought stress; p3, regulating the number of primary rachis branches and / or the number of grains per panicle and / or the yield per plant of a plant.

[0015] Wherein, the SiPYL3 protein may be the protein defined in any one of the foregoing (A1) - (A4).

[0016] Further, the regulation of the number of primary rachis branches and / or the number of grains per panicle and / or the yield per plant of a plant in p3 may be the regulation of the number of primary rachis branches and / or the number of grains per panicle and / or the yield per plant of a plant under non - stress conditions.

[0017] Further, in the said use, increasing the expression level of the SiPYL3 protein in the plant (or SiPYL3 increasing gene expression), the root length of the plant under salt stress increases and / or the shoot length increases.

[0018] Further, in the said use, increasing the expression level of the SiPYL3 protein in the plant (or SiPYL3 increasing gene expression), the survival rate of the plant under drought stress decreases.

[0019] Further, in the said use, increasing the expression level of the SiPYL3 protein in the plant (or SiPYL3 increasing gene expression), the number of primary rachis branches of the plant under non - stress conditions increases and / or the number of grains per panicle and / or the yield per plant increases.

[0020] In a third aspect, the present invention claims a method for improving the salt tolerance of a plant and / or reducing the drought resistance of a plant and / or increasing the yield of a plant.

[0021] The method for improving the salt tolerance of a plant and / or reducing the drought resistance of a plant and / or increasing the yield of a plant claimed by the present invention may include the following steps: increasing the expression level of the SiPYL3 protein in the plant (or increasing the expression level of the SiPYL3 gene in the plant), thereby achieving the improvement of the salt tolerance of the said plant and / or the reduction of the drought resistance of the said plant and / or the increase of the yield of the said plant.

[0022] In a fourth aspect, the present invention claims a method for cultivating a plant variety with specific traits.

[0023] The method for cultivating a plant variety with specific traits claimed by the present invention may include the following steps: increasing the SiPYL3The expression level of a gene (or increase the expression level of a gene in a plant), and thereby obtain a plant variety with specific traits; SiPYL3 (or increase the expression level of the gene), and then obtain a plant variety with specific traits; Among them, the specific traits may be all or part of the following traits: (1) increase in root length and / or shoot length under salt stress; (2) decrease in survival rate under drought stress; (3) increase in the number of primary branches and / or the number of grains per panicle and / or the yield per plant under non-stress conditions.

[0024] In the third and fourth aspects above, increasing the expression level of the SiPYL3 protein in the plant can be achieved by introducing the coding gene of the SiPYL3 protein into the plant (recipient plant). The improvement of plant salt tolerance, the reduction of plant drought resistance, the increase of plant yield, the increase in root length and / or shoot length under salt stress, the decrease in survival rate under drought stress, the increase in the number of primary branches and / or the number of grains per panicle and / or the yield per plant under non-stress conditions all refer to the state presented by the transgenic plant obtained after introducing the coding gene of the SiPYL3 protein compared with the recipient plant.

[0025] Furthermore, the coding gene of the SiPYL3 protein can be introduced into the plant in the form of a recombinant vector.

[0026] Even further, in the recombinant vector, the transcription of the coding gene of the SiPYL3 protein is initiated by the 35S promoter.

[0027] In an embodiment of the present invention, the recombinant vector is specifically a recombinant plasmid obtained by cloning the coding gene of the SiPYL3 protein between the restriction enzyme cleavage sites XbaI and KpnI of the pCM1307 vector.

[0028] In the above related aspects, the coding gene of the SiPYL3 protein can be any of the following: (B1) The DNA molecule shown in SEQ ID No.2 or SEQ ID No.3; (B2) A DNA molecule that hybridizes with the DNA molecule defined in (B1) under stringent conditions and encodes the SiPYL3 protein; (B3) A DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity with the DNA sequence defined in (B1) or (B2) and encodes the SiPYL3 protein.

[0029] Among the above genes, the stringent conditions may be as follows: Hybridization is carried out at 50 °C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5 M Na3PO4, and 1 mM EDTA, and rinsing is carried out at 50 °C in 2×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and rinsing is carried out at 50 °C in 1×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and rinsing is carried out at 50 °C in 0.5×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and rinsing is carried out at 50 °C in 0.1×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 50 °C in a mixed solution of 7% SDS, 0.5 M Na3PO4, and 1 mM EDTA, and rinsing is carried out at 65 °C in 0.1×SSC, 0.1% SDS; alternatively: Hybridization is carried out at 65 °C in a solution of 6×SSC, 0.5% SDS, and then the membrane is washed once with 2×SSC, 0.1% SDS and once with 1×SSC, 0.1% SDS respectively.

[0030] Among the above genes, the identity of the nucleotide sequence can be determined using identity search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastn as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of nucleotide sequences, and then the identity value (%) can be obtained.

[0031] Among the above genes, the identity of more than 95% may be at least 96%, 97%, 98% identity. The identity of more than 90% may be at least 91%, 92%, 93%, 94% identity. The identity of more than 85% may be at least 86%, 87%, 88%, 89% identity. The identity of more than 80% may be at least 81%, 82%, 83%, 84% identity.

[0032] In each of the above related aspects, the plant may be any one of the following: (C1) Monocotyledonous plants; (C2) Gramineous plants; (C3) Plants of the genus Oryza or Setaria; (C4)Rice or millet.

[0033] In one embodiment of the present invention, the plant is Zhonghua 11 rice.

[0034] Experimental results show that: the transgenic lines obtained by introducing the SiPYL3 gene from millet into rice have improved salt tolerance, reduced drought tolerance, and increased yield. This indicates that SiPYL3 the gene positively regulates plant salt tolerance and yield traits, and negatively regulates plant drought tolerance. The present invention is of great significance for cultivating salt-tolerant and high-yield plant varieties. Description of the Drawings

[0035] Figure 1 It is the gel image for positive identification of transgenic plants in Example 1. SiPYL3

[0036] Figure 2 It is the identification result of drought tolerance at the seedling stage of ZH11 and SiPYL3-OE lines in Example 2. Among them, A is the image of ZH11 and SiPYL3-OE lines under drought treatment. Scale bar, 2 cm. B is the survival rate survey of ZH11 and SiPYL3-OE lines under drought treatment. Data are mean ± SD. p-values were determined by two-tailed Student's t-test. ** p < 0.01, *** p < 0.001.

[0037] Figure 3 It is the identification result of salt tolerance at the seedling stage of ZH11 and SiPYL3-OE lines in Example 3. Among them, A is the image of ZH11 and SiPYL3-OE lines under control conditions (without salt stress treatment) and salt stress treatment. Scale bars are 5 cm (left) and 3 cm (right), respectively. B and C are the changes in root length (B) and shoot length (C) of ZH11 and SiPYL3-OE lines under control conditions and salt stress treatment, respectively. Data are mean ± SD. p-values were determined by two-tailed Student's t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0038] Figure 4 It is the yield identification result of ZH11 and SiPYL3-OE lines in Example 4. Among them, A is the phenotype of the number of primary branches of ZH11 and SiPYL3-OE lines. B is the phenotype of the number of grains per panicle of ZH11 and SiPYL3-OE lines. C is the phenotype of the yield per plant of ZH11 and SiPYL3-OE lines. Data are mean ± SD. p-values were determined by two-tailed Student's t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. Detailed Embodiments

[0039] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0040] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0041] pCM1307 vector: described in the article "Li, Gangling et al., RGN1 controls grain number and shapes panicle architecture in rice. Plant Biotechnology Journal", which can be obtained from the applicant by the public and can only be used for repeating the experiments of the present invention and cannot be used for other purposes.

[0042] Foxtail millet variety Yugu 1: "Yugu 1" described in the article "Yi, Fei et al., Time-series transcriptomics reveals a drought-responsive temporal network and crosstalk between drought stress and the circadian clock in foxtail millet. The Plant Journal, (2022), 110, 1213-1228", which can be obtained from the applicant by the public and can only be used for repeating the experiments of the present invention and cannot be used for other purposes.

[0043] Rice variety Zhonghua 11: "Zhonghua 11 (ZH11)" described in the article "Ma, Xiaoqian et al., Transcriptome analysis and identification of the low potassium stress-responsive gene SiSnRK2.6 in foxtail millet (Setaria italica L.)", which can be obtained from the applicant by the public and can only be used for repeating the experiments of the present invention and cannot be used for other purposes.

[0044] Example 1 SiPYL3 Obtaining transgenic rice I. Foxtail millet SiPYL3 Cloning of genes and cDNA Obtaining of sequences Previously, according to the results of abiotic stress, it was found that under the treatments of 20% PEG6000, 150 mM NaCl and 100 mM ABA, SiPYL3 the gene expression was significantly up-regulated. Therefore, this invention intends to conduct further functional analysis on SiPYL3 the gene. By analyzing the reference genomic sequence of Yugu 1, it was found that SiPYL3 the full-length genomic DNA of the gene is 1271 bp (SEQ ID No.2), containing 3 exons and 2 introns, and the full-length cDNA is 618 bp (SEQ ID No.3), encoding 205 amino acids (SEQ ID No.1). Then, using the cDNA reverse-transcribed from the total RNA of the roots of Yugu 1 seedlings as a template, through PCR amplification and recovery of the target fragment, it was ligated to the pEASY-Blunt Zero (TransGen Biotech Co., Ltd., Beijing) cloning vector. After successful sequencing, the cDNA sequence (SEQ ID No.3) of foxtail millet SiPYL3 was obtained.

[0045] II. SiPYL3 Obtaining of transgenic rice (1) Construction of overexpression vector The overexpression vector used in this experiment is named SiPYL3-pCM1307. The cDNA sequence (SEQ ID No.3) of foxtail millet obtained above was ligated to the 35S promoter and ligated to the plant expression vector pCM1307 by homologous recombination using recombinase (Sino-Max seamless cloning kit). The restriction enzyme sites are XbaI and KpnI, and the primers used are as follows: SiPYL3 SiPYL3-pCM1307-F: CCGTCGACGAGCTC ATGGTGGGCCTGGTCGGCG (the underlined part is the recognition sequence of the restriction enzyme site XbaI); TCTAGA SiPYL3-pCM1307-R: TTGCGGAGTACCCG TTAATGTTCGAGCGGTGAT (the underlined part is the recognition sequence of the restriction enzyme site KpnI). GGTACC The above primers are written in the 5' to 3' direction.

[0046] The above primers are written in the 5' to 3' direction.

[0047] The structure of the SiPYL3-pCM1307 vector is described as: a recombinant positive plasmid obtained by replacing the small fragment between the restriction enzyme sites XbaI and KpnI of the pCM1307 vector with the DNA fragment shown in SEQ ID No.3.

[0048] (2)Obtaining transgenic rice 2.1 Preparation of recombinant bacteria The successfully constructed overexpression vector SiPYL3-pCM1307 was transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method to obtain recombinant bacteria, named EHA105-SiPYL3-pCM1307. The callus used to infect the transgenic receptor variety was derived from rice Zhonghua 11.

[0049] 2.2 Genetic transformation of rice The classical Agrobacterium-mediated callus infection method was adopted, and the specific steps are as follows: a. Obtaining embryogenic callus: The mature seeds were dehulled, disinfected with 75% alcohol, then with 20% sodium hypochlorite solution, rinsed once with sterile water, and air-dried for 6 h. Inoculated on NB medium and cultured in the dark at 28°C for 2 weeks. The embryogenic callus was peeled off and subcultured to a new NB medium for 2 weeks of subculture.

[0050] b. Preparation of infection solution: Absorb the stored Agrobacterium tumefaciens EHA105-SiPYL3-pCM1307 solution, spread it on a solid medium containing rifampicin and kanamycin, and culture it in the dark at 28°C for 2 days in an inverted position. Scrape a small amount of Agrobacterium tumefaciens into AAM liquid medium, pipette and mix well, and measure the OD of the bacterial solution 600 to be about 0.3.

[0051] c. Co-culture: Select granular callus with natural dispersion, bright yellow color, and a diameter of about 3 - 5 mm into a triangular flask, add the prepared infection solution, infect for 15 min, absorb the excess infection solution with a sterile filter paper, and place it on a co-culture medium covered with a layer of filter paper, and co-culture at 20°C for 2 - 3 d.

[0052] d. Screening of resistant callus: Take out the co-cultured callus, wash it quickly with sterile water by shaking 5 - 6 times, then wash it with sterile water containing cephalosporin and carbenicillin for 20 min, finally place it on a sterile filter paper to drain for 3 h, and then transfer it to a delayed screening medium. After one week, transfer it to the first-round screening medium, and after two weeks, transfer it to the second-round screening medium and continue to culture for two weeks.

[0053] e. Differentiation culture: Inoculate the screened resistant callus into the pre-differentiation medium, culture it in the dark at 28°C for 2 weeks, and then transfer it to the differentiation medium for 2 - 3 weeks of light culture to obtain regenerated transgenic seedling plants.

[0054] f. Transfer the seedlings to the strong-seedling culture medium. After the seedlings take root and grow, remove them from the culture bottle, wash the culture medium off the roots, harden them for 1 - 2 weeks, and then transfer them to the field for planting until maturity.

[0055] The formula of the culture medium used in the above transgenic process is shown in Table 1.

[0056] Table 1. Formulas of various culture media used in the transgenic process Name Formulation pH value AAM liquid medium AA salts (AA macro, AA micro, iron salts) + MS vitamins + AA amino acids + 500 mg / L casein hydrolysate + 68.5 g / L sucrose + 36 g / L glucose + 20 mg / L acetosyringone 5.2 Co - culture medium Basic components of NB medium + 2 mg / L 2,4 - D + 10 g / L glucose + 20 mg / L acetosyringone 5.4 Delayed screening medium Basic components of NB medium + 2 mg / L 2,4 - D + 500 mg / L cefotaxime 5.8 First - round screening medium Basic components of NB medium + 2 mg / L 2,4 - D + 500 mg / L cefotaxime + 50 mg / L hygromycin 5.8 Second - round screening medium Basic components of NB medium + 2 mg / L 2,4 - D + 50 mg / L hygromycin 5.8 Pre - differentiation medium Basic components of NB medium + 1 mg / L 6 - BA + 2 mg / L NAA + 5 mg / L ABA + 50 mg / L hygromycin 5.8 Differentiation medium Basic components of NB medium + 2 mg / L 6 - BA + 1 mg / L NAA + 1 mg / L KT + 50 mg / L hygromycin 5.8 Seedling - strengthening medium Basic components of 1 / 2MS medium + 0.5 mg / L NAA + 0.25 mg / L paclobutrazol 5.8 Note: The basic components of the NB culture medium include N6 macroelements, B5 microelements, B5 organic components, 150 mg / L inositol, 300 mg / L hydrolyzed casein, 500 mg / L glutamine, 600 mg / L proline, 30 g / L sucrose, and 3 g / L phytagel.

[0057] (3)Identification of positive transgenic materials Identify the obtained T0 generation SiPYL3 transgenic materials at the DNA level. SiPYL3 The primers for identifying transgenic materials are: SiPYL3-pCM1307-check-F: 5’- TGCAGCAGCATACCCATAC-3’; SiPYL3-pCM1307-check-R: 5’- CCTTGATGTGCTTGACGA -3’.

[0058] The size of the target fragment is 261 bp (SiPYL3-pCM1307-check-F is a fragment on the pCM1307 vector). Plants containing the target fragment in the amplification product are positive, and plants without the target fragment are negative. The identification results of some positive samples are as Figure 1 shown.

[0059] To further clarify SiPYL3 the potential functions in response to drought stress / salt stress and in regulating plant yield, the present invention selects 3 independent homozygous overexpression lines (renamed as OE1, OE2, and OE3) from the offspring of the above-identified positive T0 generation SiPYL3 transgenic materials for subsequent functional verification.

[0060] Example 2. SiPYL3 Drought resistance identification of transgenic materials Test materials: 3 independent SiPYL3 gene homozygous overexpression lines (OE1, OE2, and OE3) obtained in Example 1, hereinafter referred to as SiPYL3-OE lines. At the same time, Zhonghua 11 (ZH11) of rice is used as a control.

[0061] To detect the drought resistance of the SiPYL3-OE line at the seedling stage, the wild-type ZH11 and overexpressing seeds were soaked in distilled water at 28-30 °C for germination for 2 days. The fully germinated seeds were selected and transplanted into soil culture boxes containing nutrient soil for growth for 15 days. Then, the water supply to the obtained seedlings was stopped for drought stress treatment until the leaves and stems withered (about 7 days). After that, rehydration was carried out for 5 days, and the survival rate was calculated. Survival was defined as having one leaf turn green. In this experiment, ZH11 and the SiPYL3-OE line were planted in the same soil culture box. At least 30 plants were evaluated in each experimental group.

[0062] The results are as Figure 2 shown. At the seedling stage, after 7 days of dehydration treatment and 5 days of recovery, the survival rate of the SiPYL3-OE line was 11.4%-38.9%, which was significantly lower than that of ZH11 (the survival rate was 64.7%). Therefore, SiPYL3 the gene negatively regulates the drought resistance of plants at the seedling stage.

[0063] The drought-sensitive rice obtained by overexpressing the SiPYL3 gene in rice in the present invention has the following specific application values: (1) Scientific research method: As a control material for studying the physiological mechanism of rice in response to drought stress, it helps to deeply analyze the physiological change process of rice in a drought environment. For example, studying the changes in physiological indexes such as photosynthesis, respiration, and water metabolism of drought-sensitive rice during drought can clarify the physiological characteristics of rice being sensitive to drought and provide a reference basis for revealing the drought tolerance mechanism of rice.

[0064] (2) In agricultural production: According to the strict water requirements of drought-sensitive rice, it can guide the rational layout of rice varieties in different regions. Plant drought-sensitive but high-quality or high-yield-potential rice varieties in areas with rich water resources to give full play to their yield and quality advantages and improve agricultural production efficiency.

[0065] (3) In the ecological environment: In some wetland ecological restoration projects, drought-sensitive rice can be used as an indicator species. Its growth status can directly reflect the water conditions and ecological environment quality of the wetland. If drought-sensitive rice grows well in the wetland, it indicates that the water and ecological environment of the wetland are more suitable, which helps to evaluate the effect of wetland ecological restoration.

[0066] Example 3, SiPYL3 Identification of the salt tolerance of transgenic materials Test materials: 3 independent SiPYL3 gene homozygous overexpressing lines (OE1, OE2, and OE3) obtained in Example 1, hereinafter referred to as the SiPYL3-OE line. At the same time, Zhonghua 11 (ZH11) in rice was used as a control.

[0067] To evaluate the salt tolerance of the SiPYL3-OE lines at the seedling stage, the seeds of the wild-type ZH11 and the overexpression lines were cultured in Hoagland nutrient solution for 10 days and then treated with 150 mM NaCl for 7 days. After that, the shoot length and root length of the ZH11 and SiPYL3-OE lines were measured respectively to evaluate their salt tolerance. The specific composition of the Hoagland nutrient solution is as follows: 0.25 mM NH4H2PO4, 1 mM CON2H4, 2 mM KCl, 1 mM MgSO4·7H2O, 1.5 mM CaCl2·H2O, 0.1 mM Fe-EDTA, 1 μΜ H3BO3, 0.05 μΜ (NH4)6Mo7O 24 .4H2O, 0.5 μΜ ZnSO4·7H2O, 1 μΜ CuSO4·5H2O, 1 μΜ MnSO4·H2O. At least 30 plants were evaluated in each experimental group. At the same time, normal growth conditions (without NaCl treatment) were set as the control.

[0068] The results are as Figure 3 shown: There was no significant difference between the SiPYL3-OE line and the ZH11 seedlings under normal growth conditions. After 7 days of treatment with 150 mM NaCl, the growth of both the ZH11 and SiPYL3-OE lines was inhibited to varying degrees. Further statistical analysis showed that the root length and shoot length of the SiPYL3-OE line were significantly higher than those of ZH11. Therefore, SiPYL3 the gene positively regulates the salt tolerance of plants at the seedling stage.

[0069] Example 4, SiPYL3 Yield identification of transgenic materials Test materials: 3 independent SiPYL3 gene homozygous overexpression lines (OE1, OE2, and OE3) obtained in Example 1, hereinafter referred to as the SiPYL3-OE lines. At the same time, rice Zhonghua 11 (ZH11) was used as the control.

[0070] In addition to abiotic stress resistance, the impact of genes on yield traits is also crucial. Therefore, the present invention further studied SiPYL3 the impact on rice panicle traits. The results are as Figure 4 shown. Compared with the wild-type ZH11, the number of primary branches, the number of grains per panicle, and the yield per plant of the three SiPYL3-OE lines all increased. Therefore, SiPYL3 the gene positively regulates the number of grains and yield of rice.

[0071] The salt-tolerant, drought-sensitive and yield-increasing SiPYL3Genetically modified rice has its unique application value. The "salt tolerance" of this type of rice enables it to survive in saline-alkali land, while the "drought sensitivity" acts like an "alarm", enabling researchers to accurately quantify its water requirements. Through this seemingly "contradictory characteristic", a scientific model for water resource management in saline-alkali land can be established (that is, through the research on this type of rice, it is clear how to precisely regulate the irrigation volume and frequency according to its drought-sensitive characteristics in saline-alkali land, avoid the aggravation of secondary salinization in saline-alkali land caused by over-irrigation, and at the same time meet the water requirements for rice growth, providing a scientific basis for the efficient utilization of water resources in rice cultivation in saline-alkali land), and ultimately achieve: (1) Water conservation: reduce ineffective irrigation and improve water use efficiency; (2) Salt control: inhibit secondary salinization and maintain soil health; (3) Yield increase: achieve stable and high yields and ensure national food security.

[0072] The above has described the present invention in detail. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In general, according to the principle of the present invention, this application intends to cover any variations, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art.

Claims

1. Use of the SiPYL3 protein or its coding gene in all or part of the following P1 - P3: P1. Regulating plant salt tolerance; P2. Regulating plant drought tolerance; P3. Regulating plant yield; The SiPYL3 protein is any one of the following: (A1) A protein with the amino acid sequence of SEQ ID No.1; (A2) A protein derived from Setaria italica that has the same function as the amino acid sequence shown in SEQ ID No.1 after substitution and / or deletion and / or addition of one or several amino acid residues; (A3) A protein derived from Setaria italica that has the same function as the amino acid sequence defined in any one of (A1) - (A2) and has an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence defined in any one of (A1) - (A2); (A4) A fusion protein obtained by linking a tag to the N - terminus and / or C - terminus of the protein defined in any one of (A1) - (A3).

2. The application according to claim 1, wherein: In the above application, increasing the expression level of the SiPYL3 protein in the plant enhances the salt tolerance of the plant; and / or In the above application, increasing the expression level of the SiPYL3 protein in the plant reduces the drought tolerance of the plant; and / or In the above application, increasing the expression level of the SiPYL3 protein in the plant increases the yield of the plant.

3. Use of increasing the expression level of the SiPYL3 protein in the plant in all or part of the following p1 - p3: p1. Regulating the root length and / or shoot length of the plant under salt stress; p2. Regulating the survival rate of the plant under drought stress; p3. Regulating the number of primary branches and / or the number of grains per panicle and / or the yield per plant of the plant; The SiPYL3 protein is any one of the following: (A1) A protein with the amino acid sequence of SEQ ID No.1; (A2) A protein derived from Setaria italica that has the same function as the amino acid sequence shown in SEQ ID No.1 after substitution and / or deletion and / or addition of one or several amino acid residues; (A3) A protein derived from Setaria italica that has the same function as the amino acid sequence defined in any one of (A1) - (A2) and has an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the amino acid sequence defined in any one of (A1) - (A2); (A4) A fusion protein obtained by linking a tag to the N - terminus and / or C - terminus of the protein defined in any one of (A1) - (A3).

4. The application according to claim 3, characterized in that: In the above application, increasing the expression level of the SiPYL3 protein in the plant increases the root length and / or shoot length of the plant under salt stress; and / or In the above application, increasing the expression level of the SiPYL3 protein in the plant reduces the survival rate of the plant under drought stress; and / or In the above application, increasing the expression level of the SiPYL3 protein in the plant increases the number of primary branches and / or the number of grains per panicle and / or the yield per plant of the plant.

5. A method for improving the salt tolerance of plants and / or reducing the drought resistance of plants and / or increasing the yield of plants, comprising the following steps: increasing the expression level of SiPYL3 protein in plants, so as to achieve improving the salt tolerance of the plants and / or reducing the drought resistance of the plants and / or increasing the yield of the plants.

6. A method for cultivating a plant variety with specific traits, comprising the following steps: increasing the expression level of SiPYL3 protein in plants, and then obtaining a plant variety with specific traits; The specific traits are all or part of the following traits: (1) increasing root length and / or shoot length under salt stress; (2) reducing the survival rate under drought stress; (3) increasing the number of primary branches and / or the number of grains per panicle and / or the yield per plant.

7. The method according to claim 5 or 6, characterized in that: Increasing the expression level of the SiPYL3 protein in the plants is achieved by introducing the coding gene of the SiPYL3 protein into the plants.

8. The method according to claim 7, wherein: The coding gene of the SiPYL3 protein is introduced into the plants in the form of a recombinant vector.

9. The application or method according to any one of claims 1-8, characterized in that: The coding gene of the SiPYL3 protein is any one of the following: (B1) The DNA molecule shown in SEQ ID No.2 or SEQ ID No.3; (B2) A DNA molecule that hybridizes with the DNA molecule defined in (B1) under stringent conditions and encodes the SiPYL3 protein; (B3) A DNA molecule that has an identity of more than 99%, more than 95%, more than 90%, more than 85% or more than 80% with the DNA sequence defined in (B1) or (B2) and encodes the SiPYL3 protein.

10. The application or method according to any one of claims 1-9, characterized in that: The plant is any one of the following: (C1) Monocotyledonous plants; (C2) Gramineous plants; (C3) Plants of the genus Oryza or Setaria; (C4) Rice or millet.

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