Application of GsSYP51b protein and its encoding gene in regulating plant stress tolerance

Abstract

The present invention discloses the use of the GsSYP51b protein and its encoding gene in regulating plant stress tolerance. The GsSYP51b protein is a1), a2), a3), or a4): a1) the amino acid sequence of the protein shown in Sequence 3; a2) a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in Sequence 3; a3) a protein related to plant stress tolerance obtained by replacing, deleting, and / or adding one or more amino acid residues in the amino acid sequence shown in Sequence 3; a4) a protein related to plant stress tolerance derived from soybean that has 90% identity to the amino acid sequence shown in Sequence 3. Experiments in the present invention demonstrate that overexpression of the GsSYP51b gene in Arabidopsis thaliana enhances its tolerance to saline-alkali stress, indicating that this protein can lay the foundation for research on breeding saline-alkali-tolerant transgenic plants.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of GsSYP51b protein and its encoding gene in regulating plant stress tolerance. Background Art

[0002] Soil salinization seriously affects crop yields and is one of the challenges facing the world in achieving sustainable development with huge economic losses. At the same time, salinization seriously restricts agricultural development in Northeast my country and even the whole country, and is a major problem facing agricultural development in my country's adverse ecological zones. According to statistics, there are 831 million hm2 of salinization in the world. 2 my country accounts for about 10% of the country's total saline-alkali land. With the continued growth of my country's population and the degradation of arable soil, the rational development and utilization of saline-alkali land and its potential for agricultural production are of great significance for increasing my country's arable land area, solving the food problem, and developing sustainable agriculture.

[0003] With the rapid development of functional genomics and molecular biology, enhancing crop salt-alkali tolerance and increasing crop yields on saline-alkali land through molecular breeding has become one of the means to improve and rationally develop and utilize saline-alkali land. However, a crucial prerequisite for achieving this goal is to identify key regulatory genes with significant functions in salt-alkali tolerance and to elucidate the salt-alkali stress signaling pathways and molecular mechanisms of salt-alkali tolerance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to regulate plant stress tolerance.

[0005] In order to solve the above technical problems, the present invention first provides a new use of the plant stress tolerance-related protein GsSYP51b.

[0006] The present invention provides the use of GsSYP51b protein in the following 1)-3):

[0007] 1) Regulate plant stress tolerance;

[0008] 2) Cultivating transgenic plants with improved stress tolerance;

[0009] 3) Plant breeding;

[0010] The GsSYP51b protein is a1) or a2) or a3) or a4):

[0011] a1) the amino acid sequence is the protein shown in SEQ ID NO: 3;

[0012] a2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 3;

[0013] a3) a protein related to plant stress tolerance obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 3;

[0014] a4) A protein having 90% identity with the amino acid sequence shown in SEQ ID NO: 3, derived from soybean and associated with plant stress tolerance.

[0015] Among them, sequence 3 consists of 251 amino acid residues.

[0016] In the protein described in a2) above, the tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and / or a SUMO tag, among others.

[0017] In the protein described in a3) above, the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues, or a substitution and / or deletion and / or addition of no more than 9 amino acid residues, or a substitution and / or deletion and / or addition of no more than 8 amino acid residues, or a substitution and / or deletion and / or addition of no more than 7 amino acid residues, or a substitution and / or deletion and / or addition of no more than 6 amino acid residues, or a substitution and / or deletion and / or addition of no more than 5 amino acid residues, or a substitution and / or deletion and / or addition of no more than 4 amino acid residues, or a substitution and / or deletion and / or addition of no more than 3 amino acid residues, or a substitution and / or deletion and / or addition of no more than 2 amino acid residues, or a substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0018] In the protein described in a4) above, the identity refers to amino acid sequence identity. Amino acid sequence identity can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, a search can be performed using blastp as the program, setting the Expect value to 10, all filters to OFF, 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. The identity value (%) can then be obtained. The identity includes amino acid sequences having 90% or greater, or 91% or greater, or 92% or greater, or 93% or greater, or 94% or greater, or 95% or greater, or 96% or greater, or 97% or greater, or 98% or greater, or 99% or greater homology to the amino acid sequence shown in SEQ ID NO: 3 of the present invention.

[0019] The protein described in a1) or a2) or a3) or a4) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0020] In order to solve the above technical problems, the present invention also provides a new use of biomaterials related to the GsSYP51b protein.

[0021] The present invention provides applications of biomaterials related to the GsSYP51b protein in the following 1)-3):

[0022] 1) Regulate plant stress tolerance;

[0023] 2) Cultivating transgenic plants with improved stress tolerance;

[0024] 3) Plant breeding.

[0025] The biological material is any one of the following A1) to A8):

[0026] A1) a nucleic acid molecule encoding the GsSYP51b protein;

[0027] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0028] A3) a recombinant vector containing the nucleic acid molecule described in A1);

[0029] A4) a recombinant vector containing the expression cassette described in A2);

[0030] A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0031] A6) a recombinant microorganism containing the expression cassette described in A2);

[0032] A7) a recombinant microorganism containing the recombinant vector described in A3);

[0033] A8) A recombinant microorganism containing the recombinant vector described in A4).

[0034] In the above application, the nucleic acid molecule described in A1) is the gene shown in B1) or B2) or B3) or B4) below:

[0035] B1) The genomic DNA molecule shown in SEQ ID NO: 1;

[0036] B2) cDNA molecule shown in sequence 2;

[0037] B3) a cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in B1) or B2) and encodes the GsSYP51b protein;

[0038] B4) a cDNA molecule or genomic DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in B1) or B2) or B3) and encodes the GsSYP51b protein.

[0039] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0040] Those skilled in the art can readily mutate the nucleotide sequence encoding the GsSYP51b protein of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequence encoding the GsSYP51b protein are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the GsSYP51b protein and have the same function.

[0041] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or greater, or 85% or greater, or 90% or greater, or 95% or greater identity to the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 3 of the present invention. Identity can be evaluated visually or with computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0042] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0043] In the above applications, the stringent conditions are hybridization and washing in a 2×SSC, 0.1% SDS solution at 68°C twice for 5 min each, and hybridization and washing in a 0.5×SSC, 0.1% SDS solution at 68°C twice for 15 min each; or hybridization and washing in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution at 65°C.

[0044] In the above application, the expression cassette (GsSYP51b gene expression cassette) containing a nucleic acid molecule encoding the GsSYP51b protein described in A2) refers to DNA capable of expressing the GsSYP51b protein in a host cell. This DNA may include not only a promoter for initiating transcription of GsSYP51b but also a terminator for terminating transcription of GsSYP51b. Furthermore, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to, constitutive promoters; tissue-, organ-, and development-specific promoters; and inducible promoters. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus (CaMV) 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators.

[0045] Existing expression vectors can be used to construct a recombinant vector containing the GsSYP51b gene expression cassette. Such plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microprojectile bombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. The plant expression vector may also contain the 3′ untranslated region of the exogenous gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3′ end of the mRNA precursor. For example, the 3′ untranslated region of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the nopaline synthase gene, Nos) and plant genes (such as the soybean storage protein gene) all have similar functions. When using gene construction plant expression vector of the present invention, also can use enhancer, comprise translation enhancer or transcription enhancer, these enhancer regions can be ATG start codon or adjacent region start codon etc., but must be identical with the reading frame of coding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and start codon is extensive, can be natural, also can be synthetic.The translation initiation region can be from transcription initiation region or structural gene. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be modified to include genes encoding enzymes or luminescent compounds that can be expressed in plants (such as the GUS gene, luciferase gene), antibiotic marker genes (such as the nptII gene, which confers resistance to kanamycin and related antibiotics; the bar gene, which confers resistance to the herbicide phosphinothricin; the hph gene, which confers resistance to the antibiotic hygromycin; the dhfr gene, which confers resistance to methotrexate; and the EPSPS gene, which confers resistance to glyphosate), chemical resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes, which provide the ability to metabolize mannose. For the safety of transgenic plants, it is possible to omit any selectable marker genes and directly screen transformed plants using stress.

[0046] In the above applications, the vector may be a plasmid, cosmid, phage or viral vector.

[0047] In the above applications, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0048] In the above application, the stress resistance may be resistance to salt and alkali stress.

[0049] Furthermore, the salt-alkali stress resistance may be carbonate stress resistance.

[0050] Furthermore, the carbonate stress resistance may be NaHCO3 stress resistance.

[0051] In the above application, the regulation of plant stress tolerance is to improve plant stress tolerance; the improvement of plant stress tolerance is manifested as follows: under NaHCO3 stress treatment, the higher the GsSYP51b protein content and / or activity in the plant or the higher the GsSYP51b gene expression level, the higher the stress tolerance of the plant; further manifested as follows: under NaHCO3 stress treatment, the higher the GsSYP51b protein content and / or activity in the plant or the higher the GsSYP51b gene expression level, the higher the seed germination rate of the plant, the higher the leaf expansion rate, the higher the fresh weight of the aboveground part (leaf fresh weight), the longer the root length, and the better the growth.

[0052] In the above application, the purpose of the plant breeding is to cultivate salt-alkali tolerant plants (such as carbonate tolerant plants).

[0053] In the above application, the plant is a monocotyledonous plant or a dicotyledonous plant, and the dicotyledonous plant can specifically be a legume and / or a cruciferous plant and / or an Asteraceae plant; the legume can be soybean, Lotus japonica, alfalfa or Pongamia chinensis; the cruciferous plant can be Arabidopsis thaliana or rapeseed; the Asteraceae plant can be sunflower; the Arabidopsis thaliana can be Arabidopsis thaliana (Columbia ecotype col-0).

[0054] In order to solve the above technical problems, the present invention finally provides a method for cultivating transgenic plants with improved stress tolerance.

[0055] The method for cultivating transgenic plants with improved stress tolerance provided by the present invention comprises the steps of increasing the content and / or activity of GsSYP51b protein in a recipient plant to obtain a transgenic plant; the transgenic plant has higher stress tolerance than the recipient plant.

[0056] In the above method, the stress resistance may be resistance to salt and alkali stress.

[0057] Furthermore, the salt-alkali stress resistance may be carbonate stress resistance.

[0058] Furthermore, the carbonate stress resistance may be NaHCO3 stress resistance.

[0059] Furthermore, the NaHCO3 stress resistance may be NaHCO3 stress resistance during the germination stage or NaHCO3 stress resistance during the seedling stage.

[0060] In a specific embodiment of the present invention, the resistance to NaHCO3 stress is specifically embodied in any one of the following X1)-X4):

[0061] X1) under carbonate stress (such as NaHCO3 stress), the seed germination rate of the transgenic plant is higher than that of the recipient plant;

[0062] X2) under carbonate stress (such as NaHCO3 stress), the leaf expansion rate of the transgenic plant is higher than that of the recipient plant;

[0063] X3) under carbonate stress (such as NaHCO3 stress), the root length of the transgenic plant is longer than that of the recipient plant;

[0064] X4) Under carbonate stress (such as NaHCO 3 stress), the aboveground fresh weight (leaf fresh weight) of the transgenic plant is higher than that of the recipient plant.

[0065] The NaHCO3 stress can specifically be 7mM NaHCO3 or 9mM NaHCO3.

[0066] In the above method, the method for increasing the content and / or activity of the GsSYP51b protein in the recipient plant is to overexpress the GsSYP51b protein in the recipient plant.

[0067] Furthermore, the overexpression method is to introduce the coding gene of the GsSYP51b protein into the recipient plant.

[0068] Furthermore, the gene encoding the GsSYP51b protein is shown in Sequence 2 in the sequence table.

[0069] In the above method, the recipient plant is a monocotyledonous plant or a dicotyledonous plant, and the dicotyledonous plant can specifically be a legume and / or a cruciferous plant and / or an Asteraceae plant; the legume can be soybean, Lotus japonica, alfalfa or Pongamia chinensis; the cruciferous plant can be Arabidopsis thaliana or rapeseed; the Asteraceae plant can be sunflower; and the Arabidopsis thaliana can be Arabidopsis thaliana (Columbia ecotype col-0).

[0070] In the above methods, the transgenic plants are understood to include not only first-generation transgenic plants obtained by transforming the GsSYP51b gene into a recipient plant, but also their progeny. Transgenic plants can be propagated within the species in which they are grown, or they can be transferred into other varieties of the same species, particularly commercial varieties, using conventional breeding techniques. Transgenic plants include seeds, callus, whole plants, and cells.

[0071] The present invention overexpresses the GsSYP51b gene in Arabidopsis thaliana to produce GsSYP51b transgenic Arabidopsis thaliana. Experiments have shown that under carbonate stress, the seed germination rate, leaf expansion rate, root length, and leaf fresh weight of the GsSYP51b transgenic Arabidopsis thaliana are higher than those of the recipient plant. This suggests that the GsSYP51b gene can enhance Arabidopsis thaliana's tolerance to salt and alkali stress, and that the GsSYP51b protein can lay the foundation for research on breeding salt and alkali-tolerant transgenic plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 To analyze the subcellular localization of GsSYP51b protein in plant cells.

[0073] Figure 2 PCR identification of GsSYP51b transgenic glufosinate-resistant seedlings.

[0074] Figure 3 RT-PCR detection of GsSYP51b transgenic Arabidopsis thaliana.

[0075] Figure 4 Analysis of carbonate tolerance during germination of GsSYP51b transgenic Arabidopsis.

[0076] Figure 5 Analysis of carbonate tolerance in GsSYP51b transgenic Arabidopsis thaliana at the seedling stage. DETAILED DESCRIPTION

[0077] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0078] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0079] The soybean material G07256 in the following examples is recorded in the literature "Ge Y, Li Y, Zhu YM, Bai X, Lv DK, Guo DJ, Ji W, Cai H: Global transcriptome profiling of wild soybean (Glycinesoja) roots under NaHCO3 treatment [J]. BMC plant biology 2010, 10." and "Ge Ying, Zhu Yanming, Lü Dekang, Dong Tingting, Wang Weishi, Tan Shangjin, Liu Caihong, Zou Ping. Study on the alkali stress response of wild soybean [J]. Grassland Science, 2009, 26 (02): 47-52." The public can obtain it from Heilongjiang Bayi Agricultural University. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0080] The pCAMBIA330035Su used in the following examples is described in the literature "Xiaoli Sun, Wei Ji, Xiaodong Ding, Xi Bai, Hua Cai, Shanshan Yang, Xue Qian, Mingzhe Sun, Yanming Zhu. GsVAMP72, novel Glycine soja R-SNARE protein, is involved in regulating plant salt tolerance and ABA sensitivity. Plant Cell Tiss Organ Cult 2013, 113: 199-215" and is publicly available from Heilongjiang Bayi Agricultural University. This biological material was used only to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0081] The pCAMBIA330035Su-NYFPu in the following examples is described in the literature "Bowei Jia, Mingzhe Sun, Huizi DuanMu, Xiaodong Ding, Beidong Liu, Yanming Zhu, Xiaoli Sun. GsCHX19.3, a member of cation / H + exchanger superfamily from wild soybean contributes to high salinity and carbonate alkaline tolerance. Sci Rep 2017, 7(1): 9423”, the public can obtain it from Heilongjiang Bayi Agricultural University. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0082] The Agrobacterium tumefaciens LBA4404 in the following examples is recorded in the document "Chen C, Sun X, Duanmu H, Zhu D, Yu Y, Cao L, Liu A, Jia B, Xiao J, Zhu Y. GsCML27, a Gene Encoding a Calcium-Binding Ef-Hand Protein from Glycine soja, Plays Differential Roles in Plant Responses to Bicarbonate, Salt and Osmotic Stresses. PLoS One. 2015 Nov 9; 10(11): e0141888. doi: 10.1371 / journal.pone.0141888. PMID: 26550992; PMCID: PMC4638360." and is available to the public from Heilongjiang Bayi Agricultural University. The biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0083] Example 1: Obtaining the Soybean Carbonate Tolerance-Related Gene GsSYP51b

[0084] 1. Download the basic information of the wild soybean SYP51b gene from the Phytozome online database. Based on the vector and gene sequence, use Primer Premier 5.0 to design gene-specific primers with Bgl II and Pac I restriction endonuclease recognition sequences as follows:

[0085] GsSYP51b-S:5'-AT AGATCT GATGCTGGCGCACTATAA-3′ (underlined is the Bgl II recognition sequence);

[0086] GsSYP51b-AS:5'-GG TTAATTAA CAAATATTTGACCAGCAGC-3' (the underlined sequence is the Pac I recognition sequence).

[0087] The above primer sequences were artificially synthesized and prepared into 100 μmol / L stock solution, used at a concentration of 10 μmol / L, and stored at -20°C.

[0088] 2. Select plump, spot-free 3-week-old wild-type soybean G07256 seedlings and use the Trizol method to extract total RNA.

[0089] 3. Synthesize the first-strand cDNA using 5×HiScript III RT SuperMix (Vazyme).

[0090] 4. Using soybean total cDNA as template, high-fidelity DNA polymerase KOD One TM PCR amplification was performed using PCR Master Mix-Blue (TOYOBO) to obtain the full-length CDS region of the GsSYP51b gene, the nucleotide sequence of which is shown in Sequence 2 in the sequence listing. The full-length CDS region of the GsSYP51b gene was ligated into the pEASY-T vector, positive clones were identified, and the clones were sent to the company for sequencing. The vector with the correct sequence was named pEASY-GsSYP51b and used for subsequent studies.

[0091] Example 2: Subcellular localization analysis of GsSYP51b protein in plant cells

[0092] 1. Based on USER TM Cloning technology was used to design the upstream primer GsSYP51b-US containing a GGCTTAAU linker and the downstream primer GsSYP51b-U-AS containing a GGTTTAAU linker. The primer sequences are as follows:

[0093] GsSYP51b-US:GGCTTAAUATGCTGGCGCACTATAAT;

[0094] GsSYP51b-U-AS: GGTTTAAUTTACAAATATTTGACCAGCAGC.

[0095] 2. Using the pEASY-GsSYP51b vector in Example 1 as a template, PCR amplification was performed using the primers in step 1 to obtain the full-length CDS region of the GsSYP51b gene.

[0096] 3. The full-length CDS region of the GsSYP51b gene obtained in step 2 was ligated with the pCAMBIA230035Su-NYFPu empty vector double-digested with Pac I and Nt.BbvC I using USER enzyme (NEB, M5505S) to construct the pCAMBIA230035Su-NYFPu-GsSYP51b vector.

[0097] 4. The pCAMBIA230035Su-NYFPu-GsSYP51b vector was transformed into Agrobacterium, and the localization of GsSYP51b protein in cells was further analyzed by transient expression in tobacco leaves. The expression position of yellow fluorescent protein was observed by laser confocal microscopy.

[0098] The results are as follows Figure 1 As shown, the results showed that NYFPu-GsSYP51b had a yellow light around the cell, that is, on the plasma membrane. Therefore, it can be inferred that the GsSYP51b protein is located in the plasma membrane.

[0099] Example 3: Obtaining GsSYP51b transgenic Arabidopsis plants and analyzing their salt-alkaline tolerance

[0100] 1. Obtaining GsSYP51b transgenic Arabidopsis plants

[0101] 1. Based on USER TM The pCAMBIA330035Su vector from the cloning technology was used as a plant overexpression vector. The pCAMBIA330035Su vector was double-digested with restriction endonucleases Pac I and Nt.BbvC I to obtain a linearized vector.

[0102] 2. Using the pEASY-GsSYP51b vector in Example 1 as a template, TM PCR amplification was performed using the primers in Example 2 under the action of PCR Master Mix-Blue (TOYOBO) high-fidelity DNA polymerase to obtain the GsSYP51b gene sequence with an HA tag at the N-terminus (HA-GsSYP51b).

[0103] 3. The obtained linearized vector, HA-GsSYP51b and USER enzyme (NEB, M5505S) were incubated at 37°C for 20 min. The uracil of the GsSYP51b gene fragment was cut with USER enzyme to form sticky ends that can complement the pCAMBIA330035Su vector. Then, the mixture was incubated at 25°C for another 20 min and transformed into Escherichia coli competent cells DH5α. The positive transformants were activated and the plasmid was extracted. The plasmid was sent to the company for sequencing analysis to ensure that there was no frameshift or mismatch during PCR amplification of the gene, thereby obtaining the recombinant expression vector pCAMBIA330035Su-GsSYP51b.

[0104] Sequencing results showed that the recombinant expression vector pCAMBIA330035Su-GsSYP51b was obtained by inserting the DNA molecule shown in sequence 2 between the two PacI restriction sites of the pCAMBIA330035Su vector, while keeping the other sequences of the pCAMBIA330035Su vector unchanged.

[0105] 4. The recombinant expression vector pCAMBIA330035Su-GsSYP51b was transformed into Agrobacterium tumefaciens LBA4404 by freeze-thaw method. The colony PCR identification was positive, indicating that the recombinant vector was successfully transformed into Agrobacterium, and the positive recombinant bacteria were recorded as pCAMBIA330035Su-GsSYP51b / LBA4404.

[0106] 5. Adopt the inflorescence infection method of Agrobacterium-mediated that recombinant bacteria pCAMBIA330035Su-GsSYP51b / LBA4404 is infected wild-type Arabidopsis thaliana (Colombia ecotype), make GsSYP51b gene overexpression in Arabidopsis thaliana.After the T0 of above-mentioned results is sterilized through NaClO, plant on the 1 / 2MS solid screening medium containing 25mg / L glufosinate-ammonium.The overexpression transgenic Arabidopsis thaliana that filters out has glufosinate-ammonium resistance is transferred in soil and cultivated, and is labeled as different strains. When gathering in the crops, individual plant single harvest.

[0107] 6. Genomic DNA from wild-type Arabidopsis thaliana (WT) and transgenic Arabidopsis thaliana expressing glufosinate resistance was extracted using the EasyPure Genomic DNA Extraction Kit from Quanshijin. PCR was performed using the specific primers used for the full-length GsSYP51b gene clone. ddH2O was used as a negative control, and the positive plasmid pCAMBIA330035Su-GsSYP51b was used as a positive control.

[0108] The results are as follows Figure 2 As shown, the results showed that the negative controls using ddH2O as a template and wild-type Arabidopsis as a template had no amplified bands, while the other samples amplified bands with the same size as the positive plasmid, indicating that the exogenous gene GsSYP51b had been integrated into the Arabidopsis genome.

[0109] 7. Each T0 generation positive plant obtained in step 6 was multiplied, and after repeated screening for 2-3 generations, a transgenic Arabidopsis homozygous strain with glufosinate resistance was obtained. Total RNA was extracted from wild-type Arabidopsis thaliana (WT) and homozygous transgenic Arabidopsis thaliana strains, and RT-PCR was performed using specific primers using a 5-fold dilution of the reverse transcription product as a template, using the Actin2 gene as an internal reference.

[0110] The results are as follows Figure 3 The results showed that, while the Actin2 gene amplification band showed consistent brightness, no amplification band was observed using WT plant cDNA as a template. However, all homozygous GsSYP51b transgenic Arabidopsis lines were able to amplify the target band, and the band size was consistent. This indicates that the exogenous gene GsSYP51b has not only been successfully integrated into the Arabidopsis genome but is also transcribed and expressed normally in the transgenic Arabidopsis. Therefore, these lines can be used in subsequent experiments. T3 generation GsSYP51b transgenic Arabidopsis homozygous lines #2, #4, and #6 were selected for the following carbonate tolerance analysis.

[0111] 2. Analysis of Carbonate Tolerance in GsSYP51b Transgenic Arabidopsis

[0112] 1. Analysis of carbonate tolerance during germination of GsSYP51b transgenic Arabidopsis

[0113] Seeds of plump and uniformly vigorous wild-type Arabidopsis (WT) and T3-generation GsSYP51b transgenic Arabidopsis lines #2, #4, and #6 were sterilized with 5% NaClO for 5 minutes under sterile conditions, rinsed repeatedly with sterile ddH₂O to remove the NaClO, and placed in a 4°C refrigerator for vernalization for 2 days. The seeds were then sown on normal 1 / 2 MS medium or 1 / 2 MS medium supplemented with 7 mM or 9 mM NaHCO₃, respectively, and incubated at 22°C for 10-14 days. Seedling growth was observed, and seed germination rates within 0-6 days were calculated. Leaf expansion rates were calculated after 14 days. The experiment was replicated three times, with 30 plants per treatment and per line used.

[0114] The results are as follows Figure 4 The results showed that: in normal 1 / 2MS medium, all lines germinated and grew normally, with no difference in germination rate and seedling growth. However, after NaHCO3 treatment, seed germination of both wild-type and GsSYP51b transgenic Arabidopsis lines was inhibited, but the seed germination rate of GsSYP51b transgenic Arabidopsis lines was higher than that of wild-type Arabidopsis. In particular, after 14 days of NaHCO3 stress treatment, the growth of all plants was inhibited compared with plants grown under normal conditions, but the leaf expansion rate of GsSYP51b transgenic Arabidopsis lines was significantly higher than that of wild-type Arabidopsis, indicating that GsSYP51b transgenic Arabidopsis lines have a higher ability to tolerate carbonate stress during the germination period than wild-type Arabidopsis.

[0115] 2. Analysis of carbonate tolerance in GsSYP51b transgenic Arabidopsis thaliana seedlings

[0116] Seeds of wild-type Arabidopsis thaliana (WT) and T3-generation GsSYP51b transgenic lines #2, #4, and #6, sterilized with NaClO, were sown on normal 1 / 2 MS solid medium and cultured for 6 days. Arabidopsis seedlings of similar growth were then transplanted vertically onto normal 1 / 2 MS medium and 1 / 2 MS medium supplemented with 7 mM NaHCO₃ for 6-10 days. Root length and aboveground fresh weight (leaf fresh weight) were measured after 10 days of culture. The experiment was replicated three times, with 30 plants per line and treatment.

[0117] The results are as follows Figure 5 The results showed that after NaHCO3 treatment, the growth of all plants was inhibited, while the seedlings of GsSYP51b transgenic Arabidopsis lines grew significantly better than those of wild-type Arabidopsis, and the root length and aboveground fresh weight (leaf fresh weight) were significantly higher than those of wild-type Arabidopsis, indicating that GsSYP51b transgenic Arabidopsis lines had a higher ability to tolerate carbonate stress than wild-type Arabidopsis during the seedling stage.

[0118] In summary, overexpression of the GsSYP51b gene significantly improved the carbonate stress tolerance of Arabidopsis thaliana, and the GsSYP51b protein can positively regulate plant salt and alkali tolerance.

[0119] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of GsSYP51b protein in the following 1) or 2): 1) Regulate plant stress tolerance; 2) Cultivate transgenic plants with improved stress tolerance; The GsSYP51b protein is a1) or a2): a1) The amino acid sequence is the protein shown in SEQ ID NO: 3; a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 3; The stress resistance is resistance to salt and alkali stress; The plant is Arabidopsis thaliana or soybean.

2. Use of the biomaterial related to the GsSYP51b protein according to claim 1 in the following 1) or 2): 1) Regulate plant stress tolerance; 2) Cultivate transgenic plants with improved stress tolerance; The biological material is any one of the following A1) to A8): A1) a nucleic acid molecule encoding the GsSYP51b protein according to claim 1; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1); A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule described in A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) a recombinant microorganism containing the recombinant vector described in A3); A8) a recombinant microorganism containing the recombinant vector described in A4); The stress resistance is resistance to salt and alkali stress; The plant is Arabidopsis thaliana or soybean.

3. The use according to claim 2, characterized in that: A1) The nucleic acid molecule is the gene shown in B1) or B2) below: B1) The genomic DNA molecule shown in SEQ ID NO:1; B2) The cDNA molecule shown in sequence 2.

4. A method for cultivating a transgenic plant with improved stress tolerance, comprising the steps of: increasing the content of the GsSYP51b protein of claim 1 in a recipient plant to obtain a transgenic plant; wherein the transgenic plant has higher stress tolerance than the recipient plant; The stress resistance is resistance to salt and alkali stress; The plant is Arabidopsis thaliana or soybean.

5. The method according to claim 4, characterized in that: The stress tolerance of the transgenic plant is higher than that of the recipient plant, which is specifically embodied in any one of the following X1) to X4): X1) under carbonate stress, the seed germination rate of the transgenic plant is higher than that of the recipient plant; X2) under carbonate stress, the leaf expansion rate of the transgenic plant is higher than that of the recipient plant; X3) under carbonate stress, the root length of the transgenic plant is longer than that of the recipient plant; X4) Under carbonate stress, the aboveground fresh weight of the transgenic plant is higher than that of the recipient plant.

6. The method according to claim 4, characterized in that: The method for increasing the content of the GsSYP51b protein according to claim 1 in the recipient plant is to overexpress the GsSYP51b protein in the recipient plant.

7. The method according to claim 6, characterized in that: The overexpression method is to introduce the coding gene of the GsSYP51b protein into the recipient plant.

8. The method according to any one of claims 4 to 7, characterized in that: The coding gene sequence of the GsSYP51b protein is shown in Sequence 2 in the sequence table.

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