Application of GsSYP71b protein or its related biological materials in cultivating salt-tolerant plants

Abstract

The present invention discloses the application of the GsSYP71b protein or its related biological materials in cultivating salt-tolerant and alkali-tolerant plants. The GsSYP71b protein of the present invention is a1) or a2) or a3) or a4): a1) a protein with the amino acid sequence shown in Sequence 3; a2) a fusion protein obtained by connecting 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 substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in Sequence 3; a4) a protein with 90% identity to the amino acid sequence shown in Sequence 3, derived from soybean and related to plant stress tolerance. The experiments of the present invention demonstrate that overexpressing the GsSYP71b gene in Arabidopsis thaliana can enhance the tolerance of Arabidopsis thaliana to carbonate stress, indicating that this protein can lay a foundation for the research on cultivating transgenic salt-tolerant and alkali-tolerant plants.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of GsSYP71b protein or related biological materials in cultivating salt-alkali tolerant plants. Background Art

[0002] According to statistics, there are 831 million hm2 in the world 2 Soil cannot be effectively utilized due to salinization. The soil in the Songnen Plain in Northeast my country is seriously salinized, with a saline-alkali area of ​​approximately 3.73 million hm2. 2 , one of the three largest saline-alkali lands in the world. Land salinization severely hinders crop growth and development, reducing crop quality and yield, and is a major issue facing sustainable agricultural development. Therefore, targeted efforts to improve crop salt-alkali tolerance are essential.

[0003] With the rapid development of molecular biology, breeding new salt- and alkali-tolerant crop varieties through molecular design is an effective means of developing and utilizing saline-alkali land and improving crop yield and quality. However, this requires identifying key regulatory genes for salt- and alkali-tolerant plants and analyzing their molecular mechanisms of stress 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 GsSYP71b.

[0006] The present invention provides the use of GsSYP71b 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 GsSYP71b 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 265 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.

[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 new uses of biomaterials related to the GsSYP71b protein.

[0021] The present invention provides applications of biomaterials related to the GsSYP71b 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) Nucleic acid molecule encoding GsSYP71b 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 SEQ ID NO: 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 GsSYP71b protein;

[0038] B4) a cDNA molecule or genomic DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in B1) or B2) or B3) and encodes the above-mentioned GsSYP71b 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 GsSYP71b 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 GsSYP71b protein are derived from and equivalent to the nucleotide sequence of the present invention, as long as they encode the GsSYP71b 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 (GsSYP71b gene expression cassette) containing a nucleic acid molecule encoding the GsSYP71b protein described in A2) refers to DNA capable of expressing the GsSYP71b protein in a host cell. This DNA may include not only a promoter for initiating transcription of GsSYP71b but also a terminator for terminating transcription of GsSYP71b. 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] Available expression vectors can be used to construct recombinant vectors containing the GsSYP71b gene expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb. The plant expression vectors can also include the 3' untranslated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as Agrobacterium crown gall induction (Ti) plasmid genes (such as the nopaline synthase gene Nos) and plant genes (such as the soybean storage protein gene) 3' untranslated regions that 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 specifically be NaHCO3 stress resistance.

[0051] In the above application, the regulation of plant stress tolerance can be to improve plant stress tolerance; the improvement of plant stress tolerance is specifically manifested as follows: under NaHCO3 stress treatment, the higher the GsSYP71b protein content and / or activity in the plant, or the higher the GsSYP71b gene expression level, the higher the stress tolerance of the plant; further manifested as follows: under NaHCO3 stress treatment, the higher the GsSYP71b protein content and / or activity in the plant, or the higher the GsSYP71b gene expression level, the higher the seed germination rate, the higher the survival rate, the longer the root length, and the better the growth of the plant.

[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 GsSYP71b 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. The NaHCO3 stress resistance may specifically be NaHCO3 stress resistance during the germination stage or NaHCO3 stress resistance during the seedling stage.

[0059] Furthermore, the NaHCO3 stress resistance may be resistance to 6 mM NaHCO3 stress.

[0060] 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):

[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 survival 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 NaHCO3 stress), the transgenic plant grows better than the recipient plant.

[0065] In the above method, the method for increasing the content and / or activity of the GsSYP71b protein in the recipient plant is to overexpress the GsSYP71b protein in the recipient plant.

[0066] Furthermore, the overexpression method is to introduce the coding gene of the GsSYP71b protein into the recipient plant.

[0067] Furthermore, the gene encoding the GsSYP71b protein is shown in Sequence 2 in the sequence table.

[0068] 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).

[0069] In the above methods, the transgenic plants are understood to include not only first-generation transgenic plants obtained by transforming the GsSYP71b 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.

[0070] The present invention overexpresses the GsSYP71b gene in Arabidopsis thaliana to produce GsSYP71b transgenic Arabidopsis thaliana. Experiments have shown that under carbonate stress, the seed germination rate, survival rate, root length, and fresh weight of the GsSYP71b transgenic Arabidopsis thaliana were higher than those of the recipient plants. This suggests that the GsSYP71b gene can enhance Arabidopsis thaliana's tolerance to carbonate stress and that the GsSYP71b protein can lay the foundation for research into the cultivation of salt- and alkali-tolerant transgenic plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Subcellular localization analysis of GsSYP71b protein.

[0072] Figure 2 PCR identification of GsSYP71b transgenic glufosinate-resistant seedlings.

[0073] Figure 3 RT-PCR detection of GsSYP71b transgenic Arabidopsis thaliana.

[0074] Figure 4 Phenotypic analysis of GsSYP71b transgenic lines during germination under saline-alkali stress. Figure A shows the phenotypes of wild-type Arabidopsis and T3-generation GsSYP71b transgenic Arabidopsis homozygous lines #3, #5, #6, and #8 under normal culture conditions and carbonate stress. Figure B shows the survival rate of wild-type Arabidopsis and T3-generation GsSYP71b transgenic Arabidopsis homozygous lines #3, #5, #6, and #8 under normal culture conditions and carbonate stress. Figure C shows the germination rate of wild-type Arabidopsis and T3-generation GsSYP71b transgenic Arabidopsis homozygous lines #3, #5, #6, and #8 under normal culture conditions. Figure D shows the germination rate of wild-type Arabidopsis and T3-generation GsSYP71b transgenic Arabidopsis homozygous lines #3, #5, #6, and #8 under carbonate stress.

[0075] Figure 5 Phenotypic analysis of GsSYP71b transgenic lines at the seedling stage under saline-alkali stress. A shows the phenotypes of wild-type Arabidopsis and T3-generation GsSYP71b transgenic lines #3, #5, #6, and #8 under normal growth conditions and carbonate stress. B shows the root length of wild-type Arabidopsis and T3-generation GsSYP71b transgenic lines #3, #5, #6, and #8 under normal growth conditions and carbonate stress. DETAILED DESCRIPTION

[0076] 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.

[0077] 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.

[0078] The soybean material G07256 in the following examples is recorded in the literature "Y.Ge, Y.Li, Y.Zhu, X.Bai, D.Lv, D.Guo, W.Ji, H.Cai. Global transcriptome profiling of wild soybean (Glycine soja) 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. Research 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.

[0079] The vector pCAMBIA330035Su used in the following examples is described in the document "X. Sun, W. Ji, X. Ding, X. Bai, H. Cai, S. Yang, X. Qian, M. Sun, Y. 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 for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0080] PM-CFP and ER-CFP in the following examples are described in the literature "BK Nelson, X. Cai, A. Nebenführ. A multi-color set of in vivo organelle markers for colocalization studies in Arabidopsis and other plants. Plant Journal, 2007, 51: 1126-1136." and are available to the public from Heilongjiang Bayi Agricultural University. The biological materials were used only to repeat the relevant experiments of the present invention and could not be used for other purposes.

[0081] The Agrobacterium tumefaciens LBA4404 in the following examples is recorded in the document "C. Chen, X. Sun, H. Duanmu, D. Zhu, Y. Yu, L. Cao, A. Liu, B. Jia, J. Xiao, Y. Zhu. 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, 10(11): e0141888." 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.

[0082] Example 1: Obtaining the Soybean Carbonate Tolerance-Related Gene GsSYP71b

[0083] 1. Obtain basic information about the soybean SYP71b gene from the Phytozome website. Based on the vector and gene sequence, design gene-specific primers with Bgl II and Pac I restriction endonuclease recognition sequences. The primer sequences are as follows:

[0084] GsSYP71b-S:5'-AT AGATCT GATGAGCGTCATCGACATTC-3′ (underlined is the Bgl II recognition sequence);

[0085] GsSYP71b-AS:5'-GG TTAATTAA TTTCTTTAGTACGTTGTATAAGTAGGC-3' (the underlined sequence is the Pac I recognition sequence).

[0086] The above primers were artificially synthesized and prepared into 100 μmol / L stock solution, used at a concentration of 10 μmol / L, and stored at -20°C.

[0087] 2. RNA extraction kit TRIzol TM Total RNA was extracted from three-week-old wild-type soybean G07256 seedlings using Reagent.

[0088] 3. According to Invitrogen SuperScript TM ⅢReverse Transcriptase instructions for first-strand cDNA synthesis.

[0089] 4. Using soybean total cDNA as template, PrimeSTAR TM PCR amplification with HS DNA Polymerase yielded the full-length CDS region of the GsSYP71b gene, the nucleotide sequence of which is shown in Sequence 2 in the sequence listing. The full-length CDS region of the GsSYP71b 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-GsSYP71b for subsequent studies.

[0090] Example 2: Subcellular localization analysis of GsSYP71b protein in plant cells

[0091] 1. The pEASY-GsSYP71b vector obtained in Example 1 was ligated with the empty pCAMBIA1302-RFP vector after double digestion with Bgl II and Pac I to construct the GsSYP71b-RFP vector.

[0092] 2. The GsSYP71b-RFP vector was transformed into Agrobacterium, and the localization of GsSYP71b protein in cells was further analyzed by transient expression in tobacco leaves, and the expression of green fluorescent protein was observed by laser confocal microscopy.

[0093] The results are as follows Figure 1 The results show that GsSYP71b-RFP and PM-CFP colocalize at the cell periphery, namely the plasma membrane. GsSYP71b-RFP also colocalizes with ER-CFP, with distinct punctate structures appearing at the cell periphery. Furthermore, in addition to the punctate structures, distinct thread-like structures were also observed for ER-CFP. This result suggests that GsSYP71b protein is dually localized to the plasma membrane and the endoplasmic reticulum.

[0094] Example 3: Obtaining GsSYP71b transgenic Arabidopsis plants and analyzing their carbonate tolerance

[0095] 1. Obtaining GsSYP71b transgenic Arabidopsis plants

[0096] 1. Based on USER TM The pCAMBIA330035Su vector from cloning technology was used as a plant overexpression vector. PCR amplification primers were designed based on the GsSYP71b gene. GGCTTAAU was added to the 5' end of the upstream primer and GGTTTAAU was added to the 5' end of the downstream primer. The primer sequences are as follows (where U represents the USER restriction site):

[0097] GsSYP71b-US: GGCTTAAUATGTACCCATACGATGTTC;

[0098] GsSYP71b-U-AS:GGTTTAAUTCATTTCTTTAGTACGTTGTATAAGTAGG.

[0099] 2. Using the pEASY-GsSYP71b vector in Example 1 as a template, PCR amplification was performed using the primers in step 1 under the action of Pfu Turbo Cx Hot start DNA polymerase to obtain the GsSYP71b gene sequence with an HA tag at the N-terminus.

[0100] 3. The pCAMBIA330035Su vector was double-digested with restriction endonucleases Pac I and Nt.BbvC I to obtain a linearized vector. The GsSYP71b gene sequence with an HA tag at the N-terminus, the linearized vector, and USER enzyme (NEB, M5505S) were then incubated at 37°C for 20 min. The uracil of the GsSYP71b gene fragment was cut with USER enzyme to form sticky ends that can complement the pCAMBIA330035Su vector. The vector was then incubated at 25°C for another 20 min and transformed into Escherichia coli competent cells DH5α. The positive transformants were activated, the plasmids were extracted, and the plasmids were 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-GsSYP71b.

[0101] Sequencing results showed that the recombinant expression vector pCAMBIA330035Su-GsSYP71b was obtained by inserting the DNA molecule shown in sequence 2 between the two Pac I restriction sites of the pCAMBIA330035Su vector, while keeping the other sequences of the pCAMBIA330035Su vector unchanged.

[0102] 4. The recombinant expression vector pCAMBIA330035Su-GsSYP71b 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-GsSYP71b / LBA4404.

[0103] 5. The recombinant strain pCAMBIA330035Su-GsSYP71b / LBA4404 was used to infect wild-type Arabidopsis thaliana (Columbia ecotype) using the Agrobacterium-mediated inflorescence infection method to overexpress the GsSYP71b gene in Arabidopsis thaliana, obtaining T0 generation transgenic Arabidopsis thaliana. The harvested T0 generation transgenic Arabidopsis thaliana seeds were sterilized with NaClO and then planted on 1 / 2MS solid screening medium containing 25 mg / L glufosinate. The overexpressing transgenic Arabidopsis thaliana that was screened for glufosinate resistance was transferred to soil for cultivation and labeled as different strains. When harvesting the seeds, each plant was harvested individually.

[0104] 6. Genomic DNA from wild-type Arabidopsis thaliana (WT) and T1 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 GsSYP71b gene clone. ddH2O was used as a negative control, and the positive plasmid pCAMBIA330035Su-GsSYP71b was used as a positive control.

[0105] The results are as follows Figure 2 As shown, the results showed that: under the premise that no band was amplified in the negative control using ddH2O instead of template and wild-type Arabidopsis as template, a band of about 700bp was amplified in the positive control, a band of the same size as the positive control was amplified in the transgenic Arabidopsis, indicating that the GsSYP71b gene has been integrated into the Arabidopsis genome.

[0106] 7. Sow T1 seeds from each strain on 1 / 2MS screening medium containing 25 mg / L glufosinate. Transfer the glufosinate-resistant transgenic Arabidopsis to soil for cultivation. Repeat screening for 2-3 generations until homozygous strains are identified from each strain. Extract total RNA from wild-type Arabidopsis (WT) and homozygous transgenic Arabidopsis strains. Use a 5-fold dilution of the reverse transcription product as a template and perform RT-PCR using specific primers, using the Actin2 gene as an internal reference.

[0107] The results are as follows Figure 3 The results showed that, when the brightness of the Actin2 amplified bands was basically the same, PCR amplification using GsSYP71b gene-specific primers was performed. Electrophoresis analysis of the PCR products showed that no bands were amplified when wild-type Arabidopsis cDNA was used as a template, while a band of approximately 700 bp was amplified in all GsSYP71b transgenic Arabidopsis lines, indicating that GsSYP71b can be normally transcribed in transgenic Arabidopsis and can be used for subsequent experiments. T3 generation GsSYP71b transgenic Arabidopsis homozygous lines #3, #5, #6, and #8 were selected for the following carbonate tolerance analysis.

[0108] 2. Analysis of Carbonate Tolerance in GsSYP71b Transgenic Arabidopsis Plants

[0109] 1. Analysis of carbonate tolerance during germination of GsSYP71b transgenic Arabidopsis

[0110] Seeds of wild-type Arabidopsis thaliana and T3-generation GsSYP71b transgenic Arabidopsis homozygous lines #3, #5, #6, and #8, showing consistent vigor, were sterilized with 5% NaClO for 5 minutes under sterile conditions, repeatedly washed with sterile water to remove the NaClO, and refrigerated at 4°C for 2 days. Seedlings were then sown in either normal 1 / 2 MS medium or 1 / 2 MS medium supplemented with 6 mM NaHCO₃ and incubated at 22°C for 6-15 days. Seedling growth was observed, and seed germination rates were calculated within 0-7 days. Survival rates were calculated on day 15. The experiment was replicated three times, with 30 plants per line and treatment used.

[0111] The results are as follows Figure 4 Results showed that within 1-4 days after sowing, the germination rate of GsSYP71b transgenic Arabidopsis seeds treated with carbonate stress was significantly faster than that of wild-type Arabidopsis. However, after 5 days of sowing, the germination rate of both wild-type and GsSYP71b transgenic Arabidopsis seeds remained above 90%. Survival statistics showed that after carbonate stress treatment, the average survival rate of wild-type Arabidopsis was 13.3%, while the average survival rates of T3 generation homozygous GsSYP71b transgenic Arabidopsis lines #3, #5, #6, and #8 were 36.7%, 31.7%, 43.3%, and 24.2%, respectively. The survival rate of GsSYP71b transgenic Arabidopsis was significantly higher than that of wild-type Arabidopsis during subsequent growth. These results indicate that GsSYP71b transgenic Arabidopsis exhibits significantly higher carbonate stress tolerance during germination than wild-type Arabidopsis.

[0112] 2. Analysis of carbonate tolerance in GsSYP71b transgenic Arabidopsis thaliana seedlings

[0113] Seeds of wild-type Arabidopsis and T3 generation GsSYP71b transgenic Arabidopsis homozygous lines #3, #5, #6 and #8 with full and uniform vigor were sown in normal 1 / 2MS solid culture medium and cultured for 7 days. Arabidopsis with uniform growth were then transferred to normal 1 / 2MS medium or 1 / 2MS medium containing 6 mM NaHCO3. After vertical culture at 22°C for 11 days, the primary roots were observed, photographed and the length of the primary roots was counted.

[0114] The results are as follows Figure 5The results showed that carbonate stress inhibited root elongation in both wild-type and GsSYP71b transgenic Arabidopsis plants, but the root length of GsSYP71b transgenic Arabidopsis was longer than that of wild-type Arabidopsis. The average primary root length of wild-type Arabidopsis was 2.89 cm, while the average primary root lengths of T3 generation GsSYP71b transgenic Arabidopsis homozygous lines #3, #5, #6, and #8 were 3.89 cm, 3.21 cm, 4.82 cm, and 4.51 cm, respectively. These results indicate that GsSYP71b transgenic Arabidopsis has significantly higher carbonate stress tolerance than wild-type Arabidopsis during the seedling stage.

[0115] In summary, overexpression of the GsSYP71b gene significantly improved the carbonate stress tolerance of Arabidopsis thaliana, and the GsSYP71b protein can positively regulate plant salt and alkali tolerance.

[0116] 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 the GsSYP71b protein in the following 1) or 2): 1) Regulating plant stress tolerance; 2) Cultivating transgenic plants with improved stress tolerance; The GsSYP71b protein is a1) or a2): a1) A protein with the amino acid sequence shown in Sequence 3; a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in Sequence 3; The stress tolerance is salt-alkali stress tolerance; The plant is Arabidopsis thaliana or soybean.

2. Use of the biological material related to the GsSYP71b protein described in Claim 1 in the following 1) or 2): 1) Regulating plant stress tolerance; 2) Cultivating 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 GsSYP71b protein; 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 tolerance is salt-alkali stress tolerance; The plant is Arabidopsis thaliana or soybean.

3. The application according to claim 2, wherein: The nucleic acid molecule described in A1) is a gene shown in the following B1) or B2): B1) The genomic DNA molecule shown in Sequence 1; B2) The cDNA molecule shown in Sequence 2.

4. A method for cultivating transgenic plants with improved stress tolerance, comprising the following steps: increasing the content of the GsSYP71b protein described in Claim 1 in a recipient plant to obtain a transgenic plant; the stress tolerance of the transgenic plant is higher than that of the recipient plant; The stress tolerance is salt-alkali stress tolerance; The plant is Arabidopsis thaliana or soybean.

5. According to the method described in Claim 4, wherein: The fact that the stress tolerance of the transgenic plant is higher than that of the recipient plant is specifically reflected in any one of the following X1)-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 survival 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 growth vigor of the transgenic plant is better than that of the recipient plant.

6. The method according to claim 4 or 5, characterized in that: The method for increasing the content of the GsSYP71b protein described in Claim 1 in the recipient plant is to overexpress the GsSYP71b protein in the recipient plant.

7. The method according to claim 6, characterized in that: The method of overexpression is to introduce the coding gene of the GsSYP71b protein into the recipient plant.

8. The method according to claim 7, wherein: The coding gene sequence of the GsSYP71b protein is shown in Sequence 2 in the sequence listing.

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