GsbZIP43 Protein Related to Plant Stress Tolerance, and Related Biomaterials and Applications

Abstract

The present invention discloses a GsbZIP43 protein related to plant stress tolerance, and related biological materials and applications thereof. The protein GsbZIP43 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 having 90% identity with the amino acid sequence shown in Sequence 3, derived from soybean and related to plant stress tolerance. Experiments of the present invention prove that overexpressing the GsbZIP43 gene in Arabidopsis thaliana can enhance the tolerance of Arabidopsis thaliana to saline-alkali stress, indicating that this protein can lay a foundation for the research on cultivating transgenic plants with saline-alkali tolerance.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the GsbZIP43 protein related to plant stress tolerance, and related biological materials and applications thereof. Background Art

[0002] Saline-alkali stress, as the most common adverse stress environmental factor affecting crop growth and development and reducing crop yields in the world, has received wide attention. Currently, there are more than 15 billion mu of saline-alkali land in the world, and China has 1.5 billion mu of saline-alkali land, accounting for about 10% of the world's total area. Therefore, the rational development and utilization of saline-alkali land and the excavation of potential arable land resources are of special significance for increasing the land use area and improving food production. In recent years, with the development of plant genetic engineering and molecular biology, cultivating saline-alkali tolerant crops through molecular design breeding methods to improve the yield and quality of crops has become a rapid and effective molecular breeding method. Therefore, it has become particularly important to excavate related saline-alkali tolerance genes and apply them to soybean saline-alkali tolerance breeding.

[0003] The plant bZIP transcription factor family, namely the basic leucine zipper family, is one of the largest transcription factor families in plants. bZIP family transcription factors are widely involved in various biological processes of plants, such as the ABA signaling pathway, seed germination, plant flowering, and various abiotic stresses, etc. In the study of soybean bZIP transcription factors, it has been reported that the introduction of GmbZIP44, GmbZIP62, and GmbZIP78 into Arabidopsis thaliana significantly improves the salt tolerance of plants; GmbZIP123 increases the oil, glucose, fructose, and sucrose contents of transgenic Arabidopsis thaliana seeds; GmbZIP60 can be significantly induced by ABA and regulates the expression of ABA-related pathway genes; GmFDL19 can interact with FT-related genes to regulate soybean flowering. However, the related functions of GsbZIP43 have not been reported yet. Summary of the Invention

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

[0005] To solve the above technical problem, the present invention first provides a new use of the protein GsbZIP43 related to plant stress tolerance.

[0006] The present invention provides the application of the GsbZIP43 protein in the following 1)-3):

[0007] 1) Regulating plant stress tolerance;

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

[0009] 3) Plant breeding;

[0010] The GsbZIP43 protein is one of a1), a2), a3), or a4):

[0011] a1) A protein with the amino acid sequence shown in Sequence 3;

[0012] a2) A fusion protein obtained by linking a tag to the N-terminus and / or C-terminus of the protein shown in Sequence 3;

[0013] 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;

[0014] a4) A protein with 90% identity to the amino acid sequence shown in Sequence 3, derived from soybean and related to plant stress tolerance.

[0015] Among them, Sequence 3 consists of 163 amino acid residues.

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

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

[0018] In the protein described in a4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in BLAST 2.1 Advanced, by using blastp 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, the identity value (%) of a pair of amino acid sequences can be calculated. The identity includes amino acid sequences having 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher homology with the amino acid sequence shown in Sequence 3 of the present invention.

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

[0020] To solve the above technical problems, the present invention also provides a new use of a biological material related to the GsbZIP43 protein.

[0021] The present invention provides the use of a biological material related to the GsbZIP43 protein in the following 1)-3):

[0022] 1) Regulating 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 GsbZIP43 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 applications, the nucleic acid molecule described in A1) is a gene shown as any of the following B1), B2), B3), or B4):

[0035] B1) The genomic DNA molecule shown in Sequence 1;

[0036] B2) The 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 by B1) or B2) and encodes the GsbZIP43 protein;

[0038] B4) A cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence defined by B1), B2), or B3) under stringent conditions and encodes the GsbZIP43 protein.

[0039] Among them, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA, etc.

[0040] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the GsbZIP43 protein of the present invention by using known methods, such as directed evolution and site-directed mutagenesis. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence encoding the GsbZIP43 protein, as long as they encode the GsbZIP43 protein and have the same function, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0041] The term "identity" used here refers to the sequence similarity with the natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence of the protein composed of the amino acid sequence shown in Coding Sequence 3 of the present invention. Identity can be evaluated by the naked eye or 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 above identity of 75% or more can be 80%, 85%, 90%, or 95% or more.

[0043] In the above application, the stringent conditions are hybridization and membrane washing twice at 68°C for 5 minutes each time in a solution of 2×SSC and 0.1% SDS, and then hybridization and membrane washing twice at 68°C for 15 minutes each time in a solution of 0.5×SSC and 0.1% SDS; or hybridization and membrane washing under the condition of 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0044] In the above application, A2) the expression cassette containing the nucleic acid molecule encoding the GsbZIP43 protein (GsbZIP43 gene expression cassette) refers to DNA that can express the GsbZIP43 protein in a host cell. This DNA may not only include a promoter that initiates the transcription of GsbZIP43, but also include a terminator that terminates the transcription of GsbZIP43. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used 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: Agrobacterium tumefaciens nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and nopaline and octopine synthase terminator.

[0045] A recombinant vector containing the GsbZIP43 gene expression cassette can be constructed using existing expression vectors. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc., such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector may further contain the 3′ untranslated region of the foreign gene, that is, it contains 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 genes of Agrobacterium tumefaciens Ti plasmid (such as the nopaline synthase gene Nos) and the 3′ untranslated regions transcribed from plant genes (such as soybean storage protein genes) have similar functions. When constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the adjacent region start codon, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. For the convenience of identifying and screening transgenic plant cells or plants, the used plant expression vector can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants (such as the GUS gene, the luciferase gene, etc.), a marker gene for antibiotics (such as the nptII gene conferring resistance to kanamycin and related antibiotics, the bar gene conferring resistance to the herbicide phosphinothricin, the hph gene conferring resistance to the antibiotic hygromycin, and the dhfr gene conferring resistance to methotrexate, the EPSPS gene conferring resistance to glyphosate), or a marker gene for anti-chemical reagents (such as an anti-herbicide gene), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress conditions.

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

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

[0048] In the above application, the stress tolerance can be salt-alkali stress tolerance.

[0049] Furthermore, the salt-alkali stress tolerance can be carbonate stress tolerance.

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

[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: under NaHCO3 stress treatment, the higher the content and / or activity of GsbZIP43 protein or the higher the expression level of GsbZIP43 gene in the plant, the higher the stress tolerance of the plant; further manifested as: under NaHCO3 stress treatment, the higher the content and / or activity of GsbZIP43 protein or the higher the expression level of GsbZIP43 gene in the plant, the longer the root length, the higher the SOD activity, the higher the POD activity, the lower the MDA content, the higher the chlorophyll content, and the better the growth of the plant.

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

[0053] In the above application, the plant is a monocotyledon or a dicotyledon. The dicotyledon may be a leguminous plant and / or a cruciferous plant and / or a compositae plant. The leguminous plant may specifically be soybean, lotus corniculatus, alfalfa or pongamia pinnata. The cruciferous plant may specifically be Arabidopsis thaliana or rape. The compositae plant may specifically be sunflower. The Arabidopsis thaliana may specifically 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 includes the step of increasing the content and / or activity of GsbZIP43 protein in a recipient plant to obtain a transgenic plant; the stress tolerance of the transgenic plant is higher than that of the recipient plant.

[0056] In the above method, the stress tolerance may be salt-alkali stress tolerance.

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

[0058] Still further, the carbonate stress tolerance may be NaHCO3 stress tolerance.

[0059] Furthermore, the NaHCO3 stress tolerance may be NaHCO3 stress tolerance at the seedling stage or NaHCO3 stress tolerance at the adult seedling stage.

[0060] In a specific embodiment of the present invention, the NaHCO3 stress tolerance is specifically manifested in any one of the following X1)-X5):

[0061] X1) Under carbonate stress (NaHCO3 stress), the root length of the transgenic plant is longer than that of the recipient plant;

[0062] X2) Under carbonate stress (NaHCO3 stress), the SOD activity of the transgenic plant is higher than that of the recipient plant;

[0063] X3) Under carbonate stress (NaHCO3 stress), the POD activity of the transgenic plant is higher than that of the recipient plant;

[0064] X4) Under carbonate stress (NaHCO3 stress), the MDA content of the transgenic plant is lower than that of the recipient plant;

[0065] X5) Under carbonate stress (NaHCO3 stress), the chlorophyll content of the transgenic plant is higher than that of the recipient plant.

[0066] The NaHCO3 stress can specifically be 4 mM NaHCO3 or 150 mM NaHCO3.

[0067] In the above method, the method for increasing the content and / or activity of the GsbZIP43 protein in the recipient plant is to overexpress the GsbZIP43 protein in the recipient plant.

[0068] Furthermore, the method of overexpression is to introduce the coding gene of the GsbZIP43 protein into the recipient plant.

[0069] Even further, the coding gene of the GsbZIP43 protein is as shown in Sequence 2 in the Sequence Listing.

[0070] In the above method, the recipient plant is a monocotyledonous plant or a dicotyledonous plant. The dicotyledonous plant can be a leguminous plant and / or a cruciferous plant and / or a compositae plant. The leguminous plant can specifically be soybean, lotus corniculatus, alfalfa or millettia pinnata. The cruciferous plant can specifically be arabidopsis thaliana or rapeseed. The compositae plant can specifically be sunflower. The arabidopsis thaliana can specifically be arabidopsis thaliana (Columbia ecotype col-0).

[0071] In the above method, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the GsbZIP43 gene into the recipient plant, but also its progeny. For transgenic plants, the gene can be propagated in this species, or the gene can be transferred into other varieties of the same species by conventional breeding techniques, especially including commercial varieties. The transgenic plants include seeds, callus, whole plants and cells.

[0072] The GsbZIP43 gene was overexpressed in Arabidopsis thaliana to obtain GsbZIP43 transgenic Arabidopsis thaliana. Experiments have proven that under carbonate stress, the root length of GsbZIP43 transgenic Arabidopsis thaliana is longer than that of the receptor plant, and the SOD activity, POD activity, and chlorophyll content are higher than those of the receptor plant, while the MDA content is lower than that of the receptor plant. This indicates that the GsbZIP43 gene can enhance the tolerance of Arabidopsis thaliana to saline-alkali stress, and the GsbZIP43 protein can lay a foundation for the research on cultivating transgenic plants with salt and alkali tolerance. Brief Description of the Drawings

[0073] Figure 1 Screening of the GsbZIP43 gene responsive to carbonate stress in wild soybean and yeast functional verification.

[0074] Figure 2 Analysis of the transcriptional activation activity of GsbZIP43 in yeast.

[0075] Figure 3 Analysis of the subcellular localization of the GsbZIP43 protein in plant cells.

[0076] Figure 4 Analysis of the expression pattern of the GsbZIP43 gene under 50 mM carbonate stress.

[0077] Figure 5 Construction of a plant expression vector and colony PCR identification after transformation into Escherichia coli / Agrobacterium tumefaciens. Among them, M: DL15K; +: positive control; -: negative control; 1 - 2: colony PCR products.

[0078] Figure 6 Molecular biological identification of transgenic Arabidopsis thaliana. Among them, M: 2K Plus II; +: positive control (pCAMBIA3300U - GsbZIP43); -: water control; 1 - 6: transgenic plants. A: PCR detection of GsbZIP43 transgenic plants; B: RT - PCR detection of GsbZIP43 transgenic plants.

[0079] Figure 7 Analysis of the carbonate stress tolerance of GsbZIP43 transgenic Arabidopsis thaliana. A: Growth of GsbZIP43 transgenic plants under carbonate stress at the seedling stage; B: Root length of GsbZIP43 transgenic plants under carbonate stress; C: Growth of GsbZIP43 transgenic plants under carbonate stress at the adult seedling stage; D: Chlorophyll content at the adult seedling stage; E: SOD activity at the adult seedling stage; F: POD activity at the adult seedling stage; G: MDA content at the adult seedling stage. Detailed Implementation Manner

[0080] 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 and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0081] 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 instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0082] The soybean material G07256 in the following embodiments 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 (Glycine soja) roots under NaHCO3 treatment [J]. BMC plant biology 2010, 10." and "Ge Ying, Zhu Yanming, Lv Dekang, Dong Tingting, Wang Weishi, Tan Shangjin, Liu Caihong, Zou Ping. Study on the alkali stress response of wild soybean [J]. Pratacultural Science, 2009, 26(02): 47-52." The public can obtain it from Heilongjiang Bayi Agricultural University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0083] The pCAMBIA330035Su in the following embodiments is recorded in the literature "Xiaoli Sun, Wei Ji, Xiaodong Ding, Xi Bai, Hua Cai, Shanshan Yang, Xue Qian, Mingzhe Sun, Yanming Zhu. GsVAMP72, a 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". The public can obtain it from Heilongjiang Bayi Agricultural University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0084] Agrobacterium tumefaciens GV3101 in the following examples is recorded in the literature "Lee CW, et al. Agrobacterium tumefaciens promotes tumor induction by modulating pathogen defense in Arabidopsis thaliana, Plant Cell, 2009, 21(9), 2948-62". The public can obtain it from Heilongjiang Bayi Agricultural University. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0085] Example 1. Obtaining of the carbonate tolerance-related gene GsbZIP43 in wild soybean

[0086] I. Screening of the GsbZIP43 gene responsive to carbonate stress in wild soybean

[0087] In the present invention, 345 wild soybean materials were collected from the severely saline-alkali land (pH = 10.6) in western Jilin. After carbonate stress treatment (150 mM NaHCO3, pH 9.5) and physiological index analysis, one strain G07256 with the strongest carbonate tolerance was screened out. Using the wild soybean G07256 with extremely strong alkali tolerance as the test material, a wild soybean cDNA library was constructed and transferred into the AH109 yeast strain. Then, SD dropout leucine medium containing 10 mM, 11 mM, 12 mM, and 13 mM NaHCO3 was used to screen for alkali stress-responsive genes. Finally, based on the constructed wild soybean cDNA library, the key regulatory gene GsbZIP43 that significantly responds to carbonate adversity was obtained.

[0088] II. Cloning of the full-length GsbZIP43 gene

[0089] 1. The full-length sequence of the GsbZIP43 gene homologous to cultivated soybean was obtained through Phytozome using homologous cloning technology. Using this as a template, the following primers for full-length gene cloning were designed with Primer Premier 5.0:

[0090] GsbZIP43-S: 5'-GGAATTC CATATG GCTTCTCCTGGTGG-3' (the underlined part is the recognition sequence of Nde I)

[0091] GsbZIP43-AS: 5'-G GAATTC TCAATACATGATCAACATTTCATTG-3' (the underlined part is the recognition sequence of EcoR I)

[0092] Synthesize the above primer sequences and prepare a 100 μmol / L stock solution. The working concentration is 10 μmol / L and it is stored at -20 °C.

[0093] 2. Select plump wild soybean G07256 seeds, treat them with concentrated H2SO4 for 10 min, rinse with sterile water 3 - 4 times, and germinate them in the dark at 25 °C for 2 - 3 d. When the buds grow to 1 - 2 cm, transfer them to 1 / 4 Hoagland nutrient solution and culture them in an artificial climate chamber. After the seedlings grow for 21 d, treat them in Hoagland nutrient solution containing 50 mM NaHCO3 for 1 h, quickly collect samples, and extract RNA using the TRIzol method according to the steps of Thermo Fisher Scientific's TRIzol RNA Isolation Reagents.

[0094] 3. Synthesize the first strand of cDNA using OligodT as the primer. The method can be referred to the instruction manual of Invitrogen's SuperScriptTMⅢ Reverse Transcriptase.

[0095] 4. Using the total cDNA of wild soybean as the template, perform PCR amplification with PrimeSTARTM HS DNA Polymerase to obtain the full - length CDS region of the GsbZIP43 gene. Its nucleotide sequence is shown as Sequence 2 in the sequence listing. Connect the full - length CDS region of the GsbZIP43 gene with the pEASY - T vector to construct the pEASY - GsbZIP43 cloning vector.

[0096] Example 2: Analysis of the transcriptional activation activity of GsbZIP43 protein

[0097] 1. After double - digesting the pEASY - GsbZIP43 cloning vector constructed in Example 1 and the pGBKT7 empty vector with Nde I and EcoR I, ligate them to construct the BD - GsbZIP43 vector.

[0098] 2. Transform the BD - GsbZIP43 vector into Escherichia coli DH5α and use the positive clones for sequencing.

[0099] 3. Respectively transform the correctly sequenced BD - GsbZIP43 vector, negative control BD empty vector, and positive vector BD - GsbZIP67 (which has been verified to have transcriptional activation activity in yeast) into the AH109 yeast strain for transcriptional activation activity identification.

[0100] The results are as Figure 2As shown in the figure, the results show that the yeast strains transformed with GsbZIP67 and GsbZIP43 can grow normally on the SD-Trp-His defective medium, while the yeast strains transformed with the BD empty vector cannot grow normally on the SD-Trp-His defective medium. X-Gal staining was also used to prove that GsbZIP43 has transcriptional activation activity. The yeast strains transformed with GsbZIP67 and GsbZIP43 were blue, and the color of the yeast strains transformed with the BD empty vector did not change significantly.

[0101] Example 3. Subcellular localization analysis of GsbZIP43 protein in plant cells

[0102] 1. PCR amplification was performed using gene-specific primers GsbZIP43-F1 and GsbZIP43-R1 to obtain the full-length CDS region of the GsbZIP43 gene. The primer sequences are as follows:

[0103] GsbZIP43-F1: 5’-AT AGATCT GATGGCTTCTCCTGGTGG-3’; among them, the underlined part is the recognition sequence of Bgl II, and the following red G ensures no frame shift with the downstream RFP protein);

[0104] GsbZIP43-R1: 5’-GG TTAATTAA ATACATGATCAACATTTCATTGTC-3’; among them, the underlined part is the recognition sequence of PacI.

[0105] 2. The full-length CDS region of the GsbZIP43 gene obtained in step 1 was ligated with the pCAMBIA1302-RFP vector to construct the pCAMBIA1302-GsbZIP43-RFP plant expression vector.

[0106] 3. The GsbZIP43-RFP plant expression vector was transformed into Agrobacterium and injected into tobacco, and then the expression of green fluorescent protein was observed under a laser confocal microscope. At the same time, OsERF096.2-YFP was used as a positive control for nuclear localization.

[0107] The results are as Figure 3 shown. The results show that the positive control OsERF096.2-YFP is expressed in the nucleus, and GsbZIP43 is also localized in the nucleus.

[0108] Example 4. Analysis of the expression pattern of GsbZIP43 gene under alkaline stress conditions

[0109] 1. Three-week-old wild soybean G07256 seedlings were treated with 50 mM NaHCO3 carbonate stress, and root tip tissues were taken at 0 h, 1 h, 3 h, 6 h, 12 h, and 24 h, respectively.

[0110] 2. Total RNA was extracted using the TRIzol method, and cDNA was obtained by reverse transcription using the SuperScriptTM III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA).

[0111] 3. Real-time PCR was performed on an ABI 7500 fluorescence quantitative PCR instrument (Applied Biosystems, USA) using the SYBR Premix ExTaqTM II Mix (TaKaRa, Shiga, Japan) SYBR quantitative kit to detect gene expression levels. Quantitative analysis was performed using the comparative CT method (△△CT), with the GAPDH gene (Genbank accession number: DQ355800) as the internal reference and untreated samples as the reference factor. After normalization with the GAPDH gene, the change in the expression level of the GsbZIP43 gene was calculated by the 2 -△△CT method, and the relative fold change of the treated sample compared to the untreated sample was used to represent the difference.

[0112] The specific primers for the GsbZIP43 gene were 5’-ATGGGGGAACTGAGCAACA-3’ and 5’-ATTGAACAACTGAGTGGTCGTCT-3’.

[0113] The specific primers for the GAPDH gene were 5’-GACTGGTATGGCATTCCGTGT-3’ and 5’-GCCCTCTGATTCCTCCTTGA-3’.

[0114] The results of quantitative Real-time PCR were as Figure 4 shown. The results showed that after carbonate stress treatment, the expression level of the GsbZIP43 gene showed an upward trend and reached its peak after 3 h of carbonate stress treatment. After 3 h, the gene expression level showed a downward trend. This indicates that the expression of the GsbZIP43 gene is induced by carbonate stress.

[0115] Example 5. Obtaining of GsbZIP43 transgenic Arabidopsis plants and analysis of carbonate stress tolerance

[0116] I. Obtaining and identification of GsbZIP43 transgenic Arabidopsis

[0117] 1. Gene-specific primers were designed based on the GsbZIP43 gene sequence and the pCAMBIA330035Su vector. The primer sequences are as follows (where U is the USER cleavage site):

[0118] GsbZIP43-U-F: 5'-GGCTTAAUATGTACCCATACGACGTACCAG-3';

[0119] GsbZIP43-U-R: 5'-GGTTTAAUTCAATACATGATCAACATTTCATTG-3'.

[0120] 2. Using the pEASY-GsbZIP43 constructed in Example 1 as a template, PCR amplification was carried out using the primers in Step 1 to obtain the full-length GsbZIP43 gene with an HA tag at the N-terminus.

[0121] 3. The pCAMBIA330035Su vector was double digested with restriction endonucleases PacI and Nt.BbvCI to obtain the vector digestion product. The obtained vector digestion product, USER enzyme (NEB, M5505S), and the full-length GsbZIP43 gene with an HA tag at the N-terminus obtained in Step 2 were incubated at 37 °C for 20 min. The USER enzyme was used to cut at the uracil of the GsbZIP43 gene fragment to form sticky ends that could be complementary to the pCAMBIA330035Su vector. Then, it was incubated at 25 °C for 20 min and transformed into Escherichia coli competent cells DH5α (TransGen Biotech, CD201-01). Single colonies were picked for PCR identification, and the positive single colonies were sent to the company for sequencing to obtain the recombinant expression vector pCAMBIA330035Su-GsbZIP43.

[0122] The sequencing results showed that: the recombinant expression vector pCAMBIA330035Su-GsbZIP43 was a vector obtained by inserting the DNA molecule shown in Sequence 2 between the two PacI digestion sites of the pCAMBIA330035Su vector and keeping the other sequences of the pCAMBIA330035Su vector unchanged. The structural schematic diagram of the pCAMBIA330035Su-GsbZIP43 recombinant expression vector is as Figure 5 shown in A. The PCR identification results are as Figure 5 shown in B.

[0123] 4. Using the freeze-thaw method, the recombinant expression vector pCAMBIA330035Su-GsbZIP43 was transformed into Agrobacterium tumefaciens GV3101, and the recombinant Agrobacterium pCAMBIA330035Su-GsbZIP43 / GV3101 was obtained by PCR identification. The PCR identification results are as Figure 5 shown in C.

[0124] 5. The recombinant Agrobacterium pCAMBIA330035Su-GsbZIP43 / GV3101 was used to infect wild-type Arabidopsis thaliana (Columbia ecotype) by the Floral-dip method. After surface disinfection of the T0 generation seeds, they were sown on 1 / 2MS medium containing 25 mg / L of oxalis for screening. The T1 generation resistant seedlings were transplanted into nutrient pots for culture to obtain overexpression transgenic Arabidopsis thaliana resistant to oxalis, and PCR identification was performed. The identification results of some resistant plants are shown in Figure 2. Figure 6 As shown in A.

[0125] 6. Total RNA from plants identified as positive by PCR was extracted, and semi-quantitative RT-PCR was performed using the amplification primers in Example 4, with the Arabidopsis ACTIN2 gene as an internal reference, to analyze the expression level of the GsbZIP43 gene in the transgenic plants.

[0126] The results are as follows Figure 6 As shown in B. As can be seen from the figure: the wild-type Arabidopsis plant RT-PCR has no amplified product, while the GsbZIP43 transgenic Arabidopsis can amplify the target band, indicating that the exogenous gene GsbZIP43 has not only been successfully integrated into the Arabidopsis genome, but can also be normally transcribed and expressed in the transgenic Arabidopsis.

[0127] The seeds of the T1 transgenic Arabidopsis plants that were positive for RT-PCR were collected and sown on 1 / 2MS medium containing 25 mg / L sand-fixing grass for screening, and the segregation of the T2 generation was observed. This process was repeated until the T3 generation GsbZIP43 transgenic Arabidopsis homozygous strains were obtained. The T3 generation GsbZIP43 transgenic Arabidopsis homozygous strains (#10) and (#12) were selected for the following carbonate tolerance analysis.

[0128] 2. Analysis of carbonate stress tolerance in GsbZIP43 transgenic Arabidopsis

[0129] 1. Select plump wild-type Arabidopsis, T3 generation GsbZIP43 transgenic Arabidopsis homozygous strains (#10) and (#12), treat with 5% NaClO for 6-8min, rinse with sterilized ddH2O 5-7 times, vernalize at 4℃ for 3d, sow in normal 1 / 2MS medium, and culture at 22℃ for 7d. Then move the seedlings to normal 1 / 2MS medium and 1 / 2MS medium containing 4mM NaHCO3 and culture vertically for 16d. Observe the growth of plants after stress treatment, and count the root length and fresh weight of plants. The experiment was repeated three times, and 30 plants were used for each treatment and each strain.

[0130] The results are as follows Figure 7As shown in A, the results showed that carbonate stress inhibited the elongation of roots of wild-type Arabidopsis and GsbZIP43 transgenic Arabidopsis, but the root length of GsbZIP43 transgenic Arabidopsis was significantly longer than that of wild-type Arabidopsis, and the fresh weight was also heavier than that of wild-type Arabidopsis but without significant changes.

[0131] 2. Select plump seeds of wild-type Arabidopsis, T3 generation GsbZIP43 transgenic Arabidopsis homozygous strains (#10) and (#12), sow them in nutrient pots (nutrient soil: Clivia soil: vermiculite 1:1:1) after vernalization, and place them in an artificial climate incubator for culture. Select 4-week-old Arabidopsis plants with consistent growth and irrigate them with 150mM NaHCO3 (pH 8.0) solution once every 3 days for stress treatment. After 15 days of treatment, observe the growth of plants after stress treatment, and detect SOD activity, POD activity, MDA content and chlorophyll content in the leaves of wild-type Arabidopsis and GsbZIP43 transgenic Arabidopsis. The experiment was repeated three times, and 30 plants were used for each treatment and each strain. Among them, the determination method of SOD adopted the NBT (light blue tetrazolium) method in "Principles and Techniques of Plant Physiology and Biochemistry Experiments" written by Li Hesheng. The determination method of POD adopts the lignin method in "Principles and Techniques of Plant Physiological and Biochemical Experiments" written by Li Hesheng. The determination method of chlorophyll adopts the spectrophotometric method in "Principles and Techniques of Plant Physiological and Biochemical Experiments" written by Li Hesheng. The determination method of MDA adopts the TBA (thiobarbituric acid) method in "Modern Physiological Experiment Guide".

[0132] The results are as follows Figure 7 As shown in CG, the results showed that after carbonate stress treatment, both wild-type Arabidopsis and GsbZIP43 transgenic Arabidopsis gradually lost green and turned purple or even died, but the growth of GsbZIP43 transgenic Arabidopsis plants was significantly better than that of wild-type Arabidopsis ( Figure 7 C). After carbonate stress treatment, the SOD activity, POD activity and MDA content of all plants increased, but the SOD activity and POD activity of GsbZIP43 transgenic Arabidopsis were significantly higher than those of wild-type Arabidopsis, and the MDA content was significantly lower than that of wild-type Arabidopsis. The chlorophyll content was further measured, and the results showed that after carbonate stress treatment, the chlorophyll content of all strains decreased, but the chlorophyll content of GsbZIP43 transgenic Arabidopsis was significantly higher than that of wild-type Arabidopsis.

[0133] The above results show that overexpression of GsbZIP43 gene significantly improved the salt-alkali stress tolerance of Arabidopsis thaliana, and GsbZIP43 protein can positively regulate plant salt-alkali tolerance.

[0134] 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 the need for 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 short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including those that depart from the scope disclosed in this application but are made using conventional techniques known in the art. Some basic features can be applied within the scope of the following appended claims.

Claims

1. Use of the GsbZIP43 protein in the following 1) or 2): 1) Regulating plant stress tolerance; 2) Cultivating transgenic plants with improved stress tolerance; The GsbZIP43 protein is a1) or a2): a1) A protein with the amino acid sequence shown in Sequence 3; a2) A fusion protein obtained by linking 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 a biological material related to the GsbZIP43 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 GsbZIP43 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, characterized in that: 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 a transgenic plant with improved stress tolerance, comprising the following steps: increasing the content of the GsbZIP43 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)-X5): X1) Under carbonate stress, the root length of the transgenic plant is longer than that of the recipient plant; X2) Under carbonate stress, the SOD activity of the transgenic plant is higher than that of the recipient plant; X3) Under carbonate stress, the POD activity of the transgenic plant is higher than that of the recipient plant; X4) Under carbonate stress, the MDA content of the transgenic plant is lower than that of the recipient plant; X5) Under carbonate stress, the chlorophyll content of the transgenic plant is higher 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 GsbZIP43 protein described in Claim 1 in the recipient plant is overexpressing the GsbZIP43 protein in the recipient plant.

7. The method according to claim 6, wherein: The method for overexpression is introducing the coding gene of the GsbZIP43 protein into the recipient plant.

8. The method according to claim 7, characterized in that: The coding gene sequence of the GsbZIP43 protein is as shown in Sequence 2 in the sequence listing.

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