CN119661675A - Application of wheat TaSSRP1 protein and its encoding gene in regulating plant salt tolerance - Google Patents

Abstract

The present invention discloses the application of wheat TaSSRP1 protein and its encoding gene in regulating plant salt tolerance. The present invention introduces DNA molecules encoding TaSSRP1 protein into wild-type Arabidopsis and wild-type wheat, respectively, to obtain TaSSRP1 overexpressed Arabidopsis and TaSSRP1 overexpressed wheat, respectively. Experiments have shown that under salt stress, compared with wild-type Arabidopsis and wild-type wheat, the salt tolerance of TaSSRP1 overexpressed Arabidopsis and TaSSRP1 overexpressed wheat are all improved, indicating that TaSSRP1 overexpression improves the tolerance of plants to salt stress. The present invention discovers for the first time that TaSSRP1 protein and its encoding gene have the function of regulating plant salt tolerance, which is of great value for breeding salt-tolerant plant varieties.

Description

Wheat TaSSRP protein and application of coding gene thereof in regulation and control of salt tolerance of plants

Technical Field

The invention relates to the field of biotechnology, in particular to application of wheat TaSSRP protein and a coding gene thereof in regulating and controlling salt tolerance of plants.

Background

The saline-alkali soil is widely distributed in the whole world and has large area. The tendency of land salinization is exacerbated by the effects of climate, hydrology and human activity. Soil salinization seriously affects agricultural production and crop yield, and threatens global grain safety. The saline-alkali soil in China is about 15 hundred million mu, and at least 1/3 of the saline-alkali soil has development and utilization potential. The variety of saline-alkali tolerant crops is cultivated, the saline-alkali soil is fully utilized and transformed, and the effective cultivated area is increased, so that the total yield of grains is improved, and the method has important significance for guaranteeing Chinese granary and firm Chinese rice bowl.

Wheat is an important grain crop and salt stress is a major factor restricting the yield of wheat. At present, the key to guaranteeing the production of wheat is to improve the salt tolerance of the wheat. Under abiotic stress conditions, plants activate transcription factors via a variety of signaling pathways, which regulate the expression of a variety of stress response related genes, thereby enhancing stress resistance of the plants. Therefore, the cloning and transformation of the gene related to the transcription factor and the further regulation of the expression of various functional genes become an important strategy and means for improving the stress resistance of crops, and have wide application prospects. In plants, ERF transcription factors are a large transcription factor family, and the research on the action of ERF transcription factors TaSSRP is insufficient at present, and no related research on TaSSRP1 in regulating salt tolerance is found.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control plant stress tolerance.

In order to solve the technical problems, the invention firstly provides a novel application of TaSSRP protein.

The invention provides the use of TaSSRP protein in any one of the following A1) -A4):

A1 Regulating and controlling plant stress tolerance;

a2 Cultivating a stress-tolerant transgenic plant;

A3 Preparing a product for regulating and controlling plant stress tolerance;

a4 Preparing a product for cultivating a transgenic plant;

The TaSSRP protein is derived from wheat and is any one of the following B1) -B4):

B1 Amino acid sequence is a protein shown in sequence 2;

B2 A fusion protein with the same function obtained by connecting a tag to the N-terminal and/or C-terminal of the amino acid sequence shown in the sequence 2;

B3 A protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2;

B4 A protein having 80% or more identity with the amino acid sequence shown in sequence 2 and having the same function.

In the protein B2), the label refers to a polypeptide or protein which is fused and expressed together with the target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. Such tags include, but are not limited to, GST (glutathione-sulfhydryl transferase) tag protein, his6 tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein), or AviTag tag protein.

The protein according to B3) above, wherein the substitution and/or deletion and/or addition of the one or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues or a substitution and/or deletion and/or addition of not more than 9 amino acid residues or a substitution and/or deletion and/or addition of not more than 8 amino acid residues or a substitution and/or deletion and/or addition of not more than 7 amino acid residues or a substitution and/or deletion and/or addition of not more than 6 amino acid residues or a substitution and/or deletion and/or addition of not more than 5 amino acid residues or a substitution and/or deletion and/or addition of not more than 4 amino acid residues or a substitution and/or deletion and/or addition of not more than 3 amino acid residues or a substitution and/or deletion and/or addition of not more than 2 amino acid residues or a substitution and/or deletion and/or addition of not more than 1 amino acid residue.

The protein according to B4) above, wherein the identity refers to the identity of the amino acid sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, expect values are set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of amino acid sequences is searched for and calculated, and then the value (%) of identity can be obtained. Such identity includes amino acid sequences having 80% or more, or 85% or more, or 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more homology to the amino acid sequences shown in sequence 2 of the present invention.

The protein of B1), B2), B3) or B4) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

In order to solve the technical problems, the invention also provides a new application of the biological material related to TaSSRP protein.

The invention provides the use of a biomaterial related to the TaSSRP protein described above in any one of the following A1) -A4):

A1 Regulating and controlling plant stress tolerance;

a2 Cultivating a stress-tolerant transgenic plant;

A3 Preparing a product for regulating and controlling plant stress tolerance;

a4 Preparing a product for cultivating a transgenic plant;

the biomaterial is any one of the following E1) to E5):

e1 Nucleic acid molecules encoding the TaSSRP protein described above;

e2 An expression cassette comprising E1) said nucleic acid molecule;

E3 A recombinant vector comprising E1) said nucleic acid molecule, or a recombinant vector comprising E2) said expression cassette;

E4 A recombinant microorganism comprising E1) said nucleic acid molecule, or a recombinant microorganism comprising E2) said expression cassette, or a recombinant microorganism comprising E3) said recombinant vector;

e5 A recombinant host cell comprising the nucleic acid molecule of E1), or a recombinant host cell comprising the expression cassette of E2), or a recombinant host cell comprising the recombinant vector of E3).

In the above application, the nucleic acid molecule of E1) is any one of the following:

F1 A DNA molecule shown in the 21 st to 839 th positions of the sequence 1;

F2 A DNA molecule which has 75% or more identity with the nucleotide sequence defined in F1) and which encodes the TaSSRP protein described above.

The nucleotide sequence encoding TaSSRP protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 80% or more identity to the TaSSRP nucleotide sequence isolated according to the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode TaSSRP1 protein and have the same function. By identity is meant sequence similarity to a native nucleic acid sequence, including nucleotide sequences that have 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the protein consisting of the amino acid sequence set forth in coding sequence 2 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

The 75% or more identity may be 75%, 80%, 85%, 90% or more identity. The 75% identity or more may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 85% identity or more may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 90% identity or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. The 95% identity or more may be at least 95%, 96%, 97%, 98% or 99% identity.

In the above applications, the expression cassette refers to a DNA capable of expressing the TaSSRP protein described above in a host cell. The expression cassette may also include single-or double-stranded nucleic acid molecules of all regulatory sequences necessary for expression of the nucleic acid molecules of the TaSSRP protein described above. The regulatory sequences are capable of directing the expression of the TaSSRP protein described above in a suitable host cell under conditions compatible with the regulatory sequences. Such regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences include promoters and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites of the vector in order to ligate the regulatory sequences with the coding region of the nucleic acid sequence encoding the protein, a ligated regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e.a nucleic acid sequence which is recognized by the host cell in which the nucleic acid sequence is expressed. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein. The promoter may be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. The control sequence may also be a suitable transcription termination sequence, a sequence that is recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the protein. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequences may also be suitable leader sequences, i.e., untranslated regions of mRNA which are important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the host cell of choice may be used in the present invention. The regulatory sequence may also be a signal peptide coding region which codes for an amino acid sequence attached to the amino terminus of the protein and which directs the encoded protein into the cell's secretory pathway. Signal peptide coding regions that direct the expressed protein into the secretory pathway of host cells used may be used in the present invention. It may also be desirable to add regulatory sequences that regulate the expression of the protein according to the growth of the host cell.

In the above applications, the vector refers to a vector capable of carrying TaSSRP a gene into a host cell for amplification and expression, and the vector may be a cloning vector or an expression vector, including, but not limited to, plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, viral vectors (e.g., retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.).

The recombinant vector is a recombinant DNA molecule constructed by connecting TaSSRP gene and the vector in vitro. Recombinant vectors containing the TaSSRP gene or TaSSRP gene expression cassette can be constructed using existing plant expression vectors. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA Co.). The plant expression vector may also comprise the 3 ́ terminal untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylation to the 3 ́ end of the mRNA precursor, and the untranslated regions transcribed from the 3 ́ end of, for example, the Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes) all have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In the above application, the microorganism may be a bacterium, fungus, actinomycete, protozoan, algae or virus. Wherein the bacteria may be derived from Escherichia sp, erwinia sp, agrobacterium sp, flavobacterium sp, alcaligenes sp, pseudomonas sp, bacillus sp, etc., but are not limited thereto, and for example, the bacteria may be Escherichia coli ESCHERICHIA COLI, bacillus subtilis Bacillus subtilis, or Bacillus pumilus. The fungus may be a yeast, which may be from the genera Saccharomyces (e.g., saccharomyces cerevisiae Saccharomyces cerevisiae), kluyveromyces (e.g., kluyveromyces lactis Kluyveromyces lactis), pichia (e.g., pichia pastoris), schizosaccharomyces (e.g., schizosaccharomyces africana Schizosaccharomyces pombe), hansenula (e.g., hansenula polymorpha Hansenula polymorpha), etc., but is not limited thereto. The fungus may also be from Fusarium sp, rhizoctonia sp, verticillium sp, penicillium sp, aspergillus sp, cephalosporium Cephalosporium sp, etc., but is not limited thereto. The actinomycetes may be derived from Streptomyces sp, nocardia sp, micromonospora sp, neurospora Streptosporangium sp, actinoplannes sp, thermoactinomyces sp, etc., but are not limited thereto. The algae may be from Fucus sp, pantoea ACHNANTHES sp, fusarium Amphiprora sp, phaeophyllum Amphora sp, cellulose sp, astrocaryus Asteromonas sp, golden yellow algae Boekelovia sp, etc., but are not limited thereto. The virus may be rotavirus, herpes virus, influenza virus, adenovirus, etc., but is not limited thereto.

In the above applications, the host cell (also referred to as a recipient cell) may be a plant cell or an animal cell. The host cell is understood to mean not only the particular recipient cell, but also the progeny of such a cell, and such progeny may not necessarily correspond, in their entirety, to the original parent cell, but are included in the scope of the host cell, due to natural, accidental, or deliberate mutation and/or alteration. Suitable host cells are known in the art, wherein the plant cells may be, but are not limited to, plant cells such as Arabidopsis thaliana (Arabidopsis thaliana), tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., and the animal cells may be mammalian cells such as Chinese hamster ovary cells (CHO cells), african green monkey kidney cells (Vero cells), baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK 293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, NK cells, etc.), avian cells such as chicken or duck cells, amphibian cells such as Xenopus laevis (Andriasdavidianus) cells, fish cells such as grass carp, trout or catfish cells, insect cells such as Sf21 cells or Sf-9 cells, etc., but are not limited thereto.

In the present invention, the recombinant microorganism (or recombinant host cell) refers to a recombinant microorganism (or recombinant host cell) having a function changed by manipulating and modifying a gene of a microorganism of interest (or host cell of interest). Such as a recombinant microorganism (or a recombinant host cell) obtained by introducing the TaSSRP gene or the recombinant vector into a microorganism (or a host cell) of interest. The recombinant microorganism (or recombinant host cell) is understood to mean not only the particular recombinant microorganism (or recombinant host cell), but also the progeny of such a cell, and the progeny may not necessarily correspond exactly to the original parent cell, but are included in the scope of the recombinant microorganism (or recombinant host cell), due to natural, accidental, or deliberate mutations and/or alterations.

In the application, the regulation of plant stress tolerance is to improve plant stress tolerance.

Further, the stress tolerance is salt tolerance.

Further, the salt tolerance is NaCl tolerance.

In one embodiment of the invention, the regulation of plant stress tolerance is the improvement of plant salt tolerance, wherein the improvement of plant salt tolerance is specifically characterized in that the plant salt tolerance is enhanced when the TaSSRP protein content and/or activity in the plant is improved under salt stress, and the improvement of TaSSRP gene expression quantity in the plant and the improvement of plant chlorophyll content under salt stress are further characterized in that.

In another embodiment of the invention, the regulation of plant stress tolerance is the improvement of plant salt tolerance, wherein the improvement of plant salt tolerance is specifically characterized in that the plant salt tolerance is enhanced when the TaSSRP protein content and/or activity in the plant is improved under salt stress, and the regulation of plant stress tolerance is further characterized in that the TaSSRP gene expression quantity in the plant is improved under salt stress, the MDA content in plant leaves is reduced, and the proline content is improved.

In order to solve the technical problems, the invention also provides a method for cultivating the anti-reversion gene plant.

The method for cultivating the transgenic plant comprises the following steps of improving the content and/or activity of TaSSRP protein in a target plant to obtain the transgenic plant, wherein the stress tolerance of the transgenic plant is higher than that of the target plant.

In the above method, the stress tolerance is salt tolerance, specifically NaCl tolerance.

In the above method, the method for increasing the content and/or activity of TaSSRP protein in the target plant is to overexpress TaSSRP protein in the target plant. The over-expression method can be to introduce the coding gene of TaSSRP protein into target plant. Such introduction includes, but is not limited to, transfection of plant cells or tissues by conventional biological methods using Ti plasmids, ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium-mediated, and the like, and cultivation of the transfected plant cells or tissues into plants.

In one embodiment of the present invention, the TaSSRP protein-encoding gene may be introduced into a plant of interest via a recombinant vector.

In one embodiment of the present invention, the TaSSRP protein-encoding gene is introduced into the plant of interest via the recombinant vector pBI121-TaSSRP 1. The recombinant vector pBI121-TaSSRP is obtained by inserting DNA molecules shown in the 21 st-839 th positions of the sequence 1 between BamHI and SaCl enzyme cutting sites of the pBI121 vector.

In the above method, the transgenic plant has salt tolerance higher than that of the target plant, and is represented by any one of the following X1) -X3):

X1) the chlorophyll content of the transgenic plant is higher than that of the plant of interest under salt stress;

x2) the proline content of the transgenic plant is greater than that of the plant of interest under salt stress;

x3) the MDA content of the transgenic plant is less than the plant of interest under salt stress.

Further, the salt stress is NaCl stress.

Still further, the NaCl stress is 300mM NaCl.

In any of the above applications or methods, the transgenic plant comprises not only the first generation transgenic plant obtained by transforming the TaSSRP gene into a recipient plant, but also its progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

In any of the above applications or methods, the plant is a dicotyledonous plant or a monocotyledonous plant, further the monocotyledonous plant is a plant of the Gramineae family, further the plant of the Gramineae family is rice (e.g., aperture 131).

The DNA molecule for encoding TaSSRP1 protein is respectively introduced into Arabidopsis thaliana and wheat to respectively obtain TaSSRP1 over-expression Arabidopsis thaliana and TaSSRP1 over-expression wheat. Experiments prove that compared with a receptor plant, the salt tolerance of TaSSRP1 over-expressed Arabidopsis thaliana and TaSSRP1 over-expressed wheat is improved under the salt stress, which indicates that the over-expression of TaSSRP1 improves the tolerance of the plant to the salt stress. The TaSSRP protein and the coding gene thereof have the function of regulating and controlling the salt tolerance of plants, and have great value for cultivating salt-tolerant plant varieties.

Drawings

FIG. 1 shows qPCR expression analysis of wheat TaSSRP1 gene and salt tolerance identification of TaSSRP over-expressed Arabidopsis. A is the expression of wheat root, stem, leaf, ear and grain tissue. B is the responsive expression of 200mM NaCl salt stress. C is a schematic diagram of a pBI121 plant over-expression vector. D is RT-PCR analysis of TaSSRP1 over-expressed Arabidopsis (wild type Arabidopsis and TaSSRP1 over-expressed Arabidopsis leaves were collected for analysis prior to salt stress treatment). E is the growth status of Arabidopsis seedlings after 300mM NaCl salt stress treatment. Wherein, WT is wild type Arabidopsis thaliana, OE1, OE2, OE3: taSSRP1 over-expresses Arabidopsis thaliana strain. WT and TaSSRP1 over-express chlorophyll content in Arabidopsis after salt stress treatment.

FIG. 2 shows qPCR expression analysis of wheat TaSSRP gene and salt tolerance identification of TaSSRP over-expressed wheat. A is the expression level of wheat TaSSRP1 gene in wild type and TaSSRP1 over-expressed wheat (wild type and TaSSRP1 over-expressed wheat strain leaves were collected for analysis before salt stress treatment). B is the growth condition of wheat seedlings after 300mM NaCl salt stress. C is the MDA content in WT and TaSSRP.sup.1 overexpressing wheat after salt stress treatment. D is the proline content in WT and TaSSRP1 over-expressed wheat after salt stress treatment.

FIG. 3 shows TaSSRP activation of stress and antioxidant enzyme gene promoters. A is stress and antioxidant enzyme gene promoter homeopathic element analysis. B is a structure map of a reporter and an effector carrier used by the dual luciferase system. C is TaSSRP double luciferase assay of 1 activation stress and antioxidant enzyme genes. * Indicating that the difference reached a 1% level.

FIG. 4 shows TaSSRP subcellular localization, transcriptional activity assay and yeast two-hybrid screen. A is subcellular localization of wheat TaSSRP1 (wheat TaSSRP protein subcellular localization was observed under confocal laser fluorescence microscope, and wavelengths of 488nm and 633nm were used for autofluorescence of GFP and chloroplast, respectively, with a scale bar of 50 μm). Analysis of transcriptional Activity of wheat TaSSRP1 (Yeast cells carrying pGBKT7-TaSSRP1 (1-272 aa, 1-127aa, and 128-272 aa) decoy vector were cultured on SD/-Trp and SD/-Trp/-His/-Ade medium, respectively.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The wheat variety Fielder in the examples below is described in document "Wang, B., Li, L., Liu, M., Peng, D., Wei, A., Hou, B., Lei, Y.,&Li, X. (2022). TaFDL2-1A confers drought stress tolerance by promoting ABA biosynthesis, ABA responses, and ROS scavenging in transgenic wheat. The Plant journal : for cell and molecular biology, 112(3), 722–737. https://doi.org/10.1111/tpj.15975".

The pBI121 vector in the examples described below is described in literature "Yu, M., Yu, Y., Song, T., Zhang, Y., Wei, F., Cheng, J., Zhang, B.,&Zhang, X. (2022). Characterization of the voltage-dependent anion channel (VDAC) gene family in wheat (Triticum aestivumL.) and its potential mechanism in response to drought and salinity stresses.Gene,809, 146031. https://doi.org/10.1016/j.gene.2021.146031".

Wheat variety JW1 in the following examples is described in literature "Yu, Y., Song, T., Wang, Y., Zhang, M., Li, N., Yu, M., Zhang, S., Zhou, H., Guo, S., Bu, Y., Wang, T., Xiang, J.,&Zhang, X. (2023). The wheat WRKY transcription factor TaWRKY1-2D confers drought resistance in transgenic Arabidopsis and wheat (Triticum aestivum L.). International journal of biological macromolecules, 226, 1203–1217. https://doi.org/10.1016/j.ijbiomac.2022.11.234".

The plant overexpression vector pCAMBIA3301 in the examples described below is described in literature "Du, L., Huang, X., Ding, L., Wang, Z., Tang, D., Chen, B., Ao, L., Liu, Y., Kang, Z.,&Mao, H. (2023). TaERF87 and TaAKS1 synergistically regulate TaP5CS1/TaP5CR1-mediated proline biosynthesis to enhance drought tolerance in wheat. The New phytologist, 237(1), 232–250. https://doi.org/10.1111/nph.18549".

Example 1, wheat TaSSRP1 Gene cloning, tissue-specific and salt stress expression Pattern analysis

1. Wheat JW1 seed is sterilized with 70% alcohol and then washed with sterile water. Seedlings were cultivated in 1/2 intensity Hoagland solution in an illumination incubator (70% humidity, 18000Lux illumination intensity) at a temperature of 20-25℃and a light/dark period of 16h/8h to give two week old wheat seedlings.

2. Wheat seedlings of two weeks old were treated under 200mM NaCl stress, and leaves were collected after 0, 1,6, 12, 24 hours of treatment, respectively, and 0 hour of leaves were used as a gene cloning template and a stress-treated control.

3. Wheat seedlings of two weeks of age were cultivated in a greenhouse at a temperature of 15-20℃and a light/dark cycle of 16h/8h, until 7 days after the wheat had matured and flowering, and the wheat roots, stems, leaves, ears and grain tissue were collected. All samples were rapidly stored at-80 ℃ for subsequent RNA extraction.

4. And (3) taking the wheat variety JW1 seedling material treated for 0h by NaCl, extracting total RNA, and performing reverse transcription to obtain cDNA. PCR amplification was performed using primers 5'-ACGCGACCACACAGGATAAGAT-3' and 5'-TGAGAACGATCACTGGTCGCTT-3' with cDNA as template to give PCT product (TaSSRP cDNA) and sequencing the PCR product.

The sequencing result shows that the nucleotide sequence of the PCR product is shown as 1 st-868 th in sequence 1, wherein 21 st-839 th are TaSSRP st gene coding sequences, and the coding amino acid sequence is TaSSRP protein of sequence 2.

5. And (3) taking the wheat variety JW1 seedling materials with different times or different tissues treated by NaCl, extracting total RNA, performing reverse transcription to obtain cDNA, taking the cDNA as a template, and performing real-time fluorescence quantitative PCR (qRT-PCR) in a fluorescence quantitative instrument by using primers 5'-ACCTTCGACAGCGCCGAG-3' and 5'-GGAGATGAAGAGTCGGGGCT-3' to detect the expression level of the TaSSRP gene.

The results are shown in FIG. 1A and FIG. 1B, and show that wheat TaSSRP gene is highly expressed in roots and leaves and is also significantly up-regulated under salt stress, which suggests that TaSSRP1 may play a role in regulating root and leaf development and salt stress response.

Example 2, taSSRP construction of an overexpressed Arabidopsis Strain and salt tolerance analysis

1. Construction of TaSSRP A1 over-expressed Arabidopsis lines

1. The TaSSRP coding region sequence shown in the 21 st to 839 th positions of the sequence 1 is connected between BamHI and SaCl enzyme cutting sites of a plant over-expression vector pBI121 to obtain a recombinant vector pBI121-TaSSRP1. The structure of the recombinant vector pBI121-TaSSRP1 is schematically shown in FIG. 1C.

2. GV3101 Agrobacterium (CAT#: AC1001, shanghai Biotechnology Co., ltd.) was transformed with recombinant vector pBI121-TaSSRP1, and identified to obtain GV3101 Agrobacterium containing recombinant vector pBI121-TaSSRP1, which was designated as recombinant bacterium GV3101/pBI121-TaSSRP1.

3. Transferring TaSSRP gene into Arabidopsis thaliana (Columbia subtype) by using recombinant GV3101/pBI121-TaSSRP1 through an agrobacterium-mediated flowering method, harvesting T ₀ generation seeds, screening T ₀ generation seeds by using MS culture medium containing kanamycin to obtain T ₁ generation seedlings, independently transferring the T ₁ generation seedlings into matrix soil for culture until the seedlings are mature, harvesting the T ₂ generation seeds, screening the T ₂ generation seeds by using MS culture medium containing kanamycin, and selecting resistant (green seedlings) and sensitive (albino seedlings) T ₂ generation resistant seedlings with a separation ratio of 3:1.

T ₀ represents the Arabidopsis seeds harvested in the current generation after the dip-flower infestation, T ₁ represents the seeds obtained in the current generation of the transformation and the plants grown therefrom, and T ₂ represents the seeds produced by the T ₁ selfing and the plants grown therefrom.

4. Total RNAs of T ₂ generation TaSSRP1 over-expressing arabidopsis strains OE1, OE2, OE3 and wild arabidopsis were extracted and reverse transcribed into cDNA. PCR amplification was performed using primers 5'-ATGCTGCTGCTTAATCCGGC-3' and 5'-GGAGATGAAGAGTCGGGGCT-3', with the Arabidopsis thaliana Actin2 gene as the reference gene, and the primer sequences of the reference genes were as follows 5'-CTAAGCTCTCAAGATCAAAGGC-3' and 5'-AACATTGCAAAGAGTTTCAAGG-3'.

The results are shown in FIG. 1D, which shows that the reference gene can be detected in both TaSSRP over-expressed Arabidopsis lines and wild type Arabidopsis. However, the fragment of interest was detectable only in TaSSRP a1 overexpressing arabidopsis lines, but not in wild type arabidopsis.

And (3) transferring positive T ₂ generation Arabidopsis seedlings into matrix soil for culture until the seedlings are mature, obtaining T ₃ generation single copy transgenic Arabidopsis seeds, and finally selecting three T ₃ generation TaSSRP1 overexpression Arabidopsis strains OE1, OE2 and OE3 for salt tolerance analysis.

2. Salt tolerance analysis of TaSSRP A1 over-expressed Arabidopsis

The test materials are wild type Arabidopsis thaliana (WT) and T ₃ generation TaSSRP1 over-expressed Arabidopsis thaliana strains OE1, OE2 and OE3 of 40 strains each.

The experimental method comprises the steps of sterilizing seeds, sowing the seeds in an MS solid culture medium, and culturing the seeds under the conditions of 22 ℃ and 16 hours of illumination/8 hours of darkness to obtain three-week-old arabidopsis seedlings. Salt stress treatment was performed on three-week-old Arabidopsis seedlings by culturing Arabidopsis seedlings in a flowerpot filled with nutrient soil (substrate: vermiculite=3:1), at 22℃under 16 hours of light/8 hours of darkness, irrigating the pot with 300mM NaCl aqueous solution each time, 200mL of 300mM NaCl aqueous solution each time, twice a day, continuously irrigating for 13 days, photographing after the salt stress treatment for 13 days, and then measuring the total chlorophyll content (including chlorophyll a and chlorophyll b) in the leaves of each Arabidopsis strain using a Soy-Laibao chlorophyll content kit (product number: BC 0995). And normal water supply is used as a control.

The table shows that after 13 days of salt stress treatment, WT leaf blight was severe, while TaSSRP a1 overexpressing Arabidopsis lines all exhibited relatively mild symptoms (FIG. 1E). There was no difference in chlorophyll content between WT and TaSSRP1 over-expressed lines under normal water supply conditions, and total chlorophyll content in TaSSRP1 over-expressed arabidopsis lines was significantly higher than WT under salt stress conditions (fig. 1F). The above results indicate that overexpression of the wheat TaSSRP1 gene in arabidopsis can enhance salt tolerance in arabidopsis.

Examples 3, taSSRP1 construction of overexpressed wheat lines and analysis of salt tolerance

1. Construction of TaSSRP A1 over-expressed wheat Strain

1. The TaSSRP coding region sequence shown in the 21 st to 839 th positions of the sequence 1 is connected between BamHI and SpeI cleavage sites of the plant over-expression vector pCAMBIA3301 to obtain a recombinant vector pCAMBIA3301-TaSSRP1.

2. The recombinant vector pCAMBIA3301-TaSSRP was transformed into EHA105 Agrobacterium (Shanghai Biotechnology Co., ltd., CAT#: AC 1010), and identified to obtain EHA105 Agrobacterium containing the recombinant vector pCAMBIA3301-TaSSRP1, which was designated as recombinant bacterium GV3101/pCAMBIA3301-TaSSRP1.

3. The recombinant vector pCAMBIA3301-TaSSRP is transformed into young embryo callus of wheat variety Fielder by adopting an agrobacterium infection method, and three T ₃ generation transgenic wheat strains which are named OE1, OE2 and OE3 respectively are finally obtained through screening, pre-differentiation, PCR identification and generation adding.

4. In the trefoil period, wild wheat field and T ₃ generation transgenic wheat strains OE1, OE2 and OE3 are taken, total RNA is extracted and then is reversely transcribed into cDNA, and then real-time fluorescence quantitative PCR (qRT-PCR) is carried out on a fluorescence quantitative instrument by adopting primers 5'-ACCTTCGACAGCGCCGAG-3' and 5'-GGAGATGAAGAGTCGGGGCT-3', so that the expression level of TaSSRP genes is detected.

The results are shown in FIG. 2A and show that the expression levels of TaSSRP1 in the T ₃ generation TaSSRP1 transgenic wheat lines OE1, OE2 and OE3 are significantly up-regulated compared to wild-type wheat. The T ₃ generation TaSSRP1 transgenic wheat lines OE1, OE2 and OE3 were selected for the salt tolerance analysis described below.

2. Salt tolerance analysis of TaSSRP over-expressed wheat

The test materials are wild type Arabidopsis thaliana (WT), T ₃ generation TaSSRP1 over-expressed wheat strains OE1, OE2 and OE3 40 strains each.

The experimental method comprises the steps of after seed disinfection, sowing in an MS solid culture medium, culturing for 2 days under the conditions of 23 ℃ and 16 hours of light/8 hours of darkness, then transferring into a basin containing 250 g of nutrient soil, namely vermiculite=1:1, continuing to perform salt stress treatment when culturing for three hours under the conditions of 23 ℃ and 16 hours of light/8 hours of darkness and humidity of >50%, wherein the salt stress treatment method comprises the steps of transferring wheat plants into a flowerpot containing nutrient soil (substrate, namely vermiculite=3:1), culturing under the conditions of 23 ℃ and 16 hours of light/8 hours of darkness, irrigating in the basin by adopting a NaCl aqueous solution with the concentration of 300mM, irrigating 200mL of NaCl aqueous solution with the concentration of 300mM each time, continuously irrigating for 20 days, observing the phenotype after the salt stress treatment for 20 days, photographing, and measuring the MDA content and the proline content in leaves by using a Soxhlet MDA content and proline content measuring kit. And normal water supply is used as a control.

The results are shown in FIG. 2B, which shows that TaSSRP.sup.1 over-expressed wheat strain grew better than WT under salt stress treatment conditions, and that WT withered seriously. Under normal water supply conditions, there was no difference in MDA and proline levels between the WT and TaSSRP1 over-expressed wheat lines, the MDA level in TaSSRP1 over-expressed wheat line was significantly lower than WT under salt stress treatment conditions (fig. 2C), while for the proline level index, the TaSSRP1 over-expressed wheat line was significantly higher than WT (fig. 2D). The above results indicate that over-expression of TaSSRP gene in wheat can enhance wheat salt tolerance.

Example 4, taSSRP influence of 1 on stress and Activity of antioxidant Gene promoters

1. To resolve TaSSRP molecular mechanisms that regulate salt tolerance, the promoters of the wheat stress response (TaP CS1, taDREB1, taERF) and antioxidant enzymes (TaPOD, taCAT and TaSOD (Fe)) genes (2500 bp upstream of ATG) were analyzed using the PLANTCARE database and mapped for promoter DRE elements (TaSSRP 1 transcription factor binding specific elements) of these genes. As a result, as shown in FIG. 3A, it was found that the genes contained the TaSSRP 1-bound DRE element except for TaPOD gene.

2. To investigate whether TaSSRP1 was able to activate the activities of TaP5CS1, taDREB1, taERF, taPOD, taCAT and TaSOD (Fe) promoters, the promoter sequence fragments of these genes were cloned into pGreen II0800 vectors containing Renilla luciferase (REN) gene driven by CaMV35S promoter, respectively, to obtain pGreen II0800 recombinant vector （pGreen II 0800-TaP5CS1、pGreen II 0800-TaDREB1、pGreen II 0800-TaERF3、pGreen II 0800-TaPOD、pGreen II 0800-TaCAT or pGreen II 0800-TaSOD (Fe), and the TaSSRP1 CDS sequence was cloned into pGreenII-SK vector in the same manner to obtain pGreenII-SK-TaSSRP 1 recombinant vector (FIG. 3B). The pGreen II 0800-TaP5CS1、pGreen II 0800-TaDREB1、pGreen II 0800-TaERF3、pGreen II 0800-TaPOD、pGreen II 0800-TaCAT、pGreen II 0800-TaSOD（Fe）、pGreenII 62-SK-TaSSRP1 plasmids were transformed into Agrobacterium (strain GV3101 comprising pSoup-19), and the agrobacteria containing pGreen II0800 recombinant vector were mixed with Agrobacterium containing pGreenII-SK-TaSSRP 1 plasmid at a volume ratio of 1:1, centrifuged, resuspended in permeation buffer (150mm Acetosyringone,10mm MgCl ₂, 10mm MES, pH 5.6), and finally injected into Nicotiana benthamiana leaves for transient expression. The detection of the LUC and REN fluorescence ratios was performed using a microplate reader 2 days after transfection using a dual luciferase detection system, with a minimum of three biological replicates per combination.

The results are shown in FIG. 3B, which shows that TaSSRP1 was able to activate the promoter activity of the TaPOD genes, except for those genes. The above results demonstrate that TaSSRP1 may be able to activate stress and expression of antioxidant enzyme genes to regulate wheat salt tolerance.

Example 5 subcellular localization, transcriptional activation Activity and interaction protein analysis of wheat TaSSRP1

1. Subcellular localization of wheat TaSSRP1

The TaSSRP coding region sequence without termination codon was ligated to the Pcamcia 1302-GFP vector to give the recombinant vector Pcamcia 1302-GFP-TaSSRP1. The recombinant vector Pcambai 1302-GFP-TaSSRP1 is transformed into GV3101 agrobacterium strain to obtain recombinant bacteria GV 3101/Pcambai 1302-GFP-TaSSRP1. Recombinant GV 3101/Pcammia 1302-GFP-TaSSRP is injected into leaf of Nicotiana benthamiana for instantaneous expression, and recombinant GV 3101/Pcammia 1302-GFP is used as reference. GFP signal was observed after 48 hours using an olympus laser confocal microscope.

The results are shown in FIG. 4A, which shows that the fluorescent signal of TaSSRP1 is concentrated mainly in the nucleus compared to the control, indicating that TaSSRP is localized mainly in the nucleus.

2. Wheat TaSSRP A transcriptional activation Activity

The coding sequence of TaSSRP protein full length (1-272 aa), the coding sequence of TaSSRP protein N end cut-off fragment (N end 1-127 aa) and the coding sequence of TaSSRP protein C end cut-off fragment (C end 128-272 aa) are respectively connected into pGBKT7 bait vector to respectively obtain recombinant vectors pGBKT7-TaSSRP1 (1-272 aa), pGBKT7-TaSSRP1 (N end 1-127 aa) and pGBKT7-TaSSRP1 (C end 128-272 aa), and then AH109 yeast strain is transformed. To evaluate transcriptional activation activity, clones were picked up and shaken overnight at 30℃and 180rpm in SD/-Trp broth. 1.2. Mu.L of yeast solution was cultured on SD/-Trp and SD/-Trp/-His/-Ade solid media in an inverted manner for 1-3 d to observe the growth of yeast colonies, which indicated that the protein had transcriptional activation activity if yeast could grow on both media.

The results are shown in FIG. 4B, which shows that all recombinant vector transformed AH109 strains were able to grow on SD/-Trp medium, but only the N-terminal AH109 strain was able to grow on SD/-Trp/-His/-Ade medium. The above results indicate that TaSSRP protein has no transcriptional activation activity over its full length and that the transcriptional activation region is N-terminal.

3. Wheat TaSSRP yeast two-hybrid screening and verification

The plasmid containing pGBKT7-TaSSRP1 was transformed into AH109 yeast cells for further yeast two-hybrid screening. The monoclonal was picked up in a 2mL centrifuge tube (containing 1mL SD/-Trp liquid medium) and shaken overnight at 200 rpm. Then, the culture was expanded, 100. Mu.L of the culture was aspirated into 40mL of SD/-Trp liquid medium, and after culturing for 15 hours, the OD _600nm was set to about 0.8. Centrifugation at 5000rpm for 5min at normal temperature, supernatant removal, followed by resuspension of the pellet with SD/-Trp broth (4 mL). To a 2L sterile Erlenmeyer flask, 4mL of the resuspended strain, 1mL of the library strain, 45mL of 2 XYPDA were added sequentially. Slowly shaking culture is carried out for 22 hours at 30 ℃ and 50 rpm. It is then necessary to observe under an optical microscope whether yeast binders are present. If the binder appears, the next step is continued, otherwise, shake culture is needed to be continued. Centrifuge at 4500rpm for 4min, remove supernatant, then resuspend pellet with 50mL 0.5XYPDA. The centrifugation and supernatant removal operations described above were repeated, and the cells were resuspended in 10mL of 0.5 XYPDA. The resuspension bacteria liquid is coated on SD/-Trp/-His/-Leu/-Ade solid culture medium and cultured for 4d at 30 ℃. Monoclonal in SD/-Trp/-His/-Leu/-Ade liquid medium, cultured at 30 ℃ for 2d. Extracting yeast plasmid by using a root yeast plasmid extraction kit, transforming the collected plasmid into DH5 alpha escherichia coli, picking up monoclonal, shaking and sequencing. Sequencing results showed that the genes screened from the cDNA library possessed complete CDS sequences. The recombinant plasmids containing pGBKT7-TaSSRP1 and pGADT7-prey were co-transformed into AH109 yeast strains, and cultured on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade/+X-alpha-Gal solid media for gyratory verification, respectively.

As shown in FIG. 4C, the results show that AH109 yeast strains co-transferred pGADT7-TaNF-YC6 and pGBKT7-TaSSRP1 can grow on SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade/+ X-alpha-Gal media, indicating that TaSSRP1 has an interaction relationship with TaNF-YC6 candidate interacting proteins in yeast systems.

The interaction relationship between TaSSRP and TaNF-YC6 proteins was further verified in plant cells. The TaSSRP and TaNF-YC6 genes are respectively connected to pCAMBIA1302-nYFP and pCAMBIA1302-cYFP vectors to respectively obtain recombinant vectors pCAMBIA1302-nYFP-TaSSRP1 and pCAMBIA1302-cYFP-TaNF-YC6. The recombinant vectors pCAMBIA1302-nYFP-TaSSRP1 and pCAMBIA1302-cYFP-TaNF-YC6 co-transform Agrobacterium GV3101 and transiently co-express in tobacco. After 48h YFP fluorescence signal was observed using a laser confocal microscope. If a YFP fluorescent signal is present, it is stated that TaSSRP and TaNF-YC6 proteins interact in tobacco.

The results are shown in FIG. 4D, which shows that further validation of the interaction relationship between TaSSRP and TaNF-YC6 with BiFC, YFP fluorescence was observed when TaSSRP and TaNF-YC6 co-transferred into tobacco, whereas no YFP fluorescence was observed in the control combination, indicating the interaction between TaSSRP and TaNF-YC6 in tobacco cells.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1. Use of the protein in any of the following A1)-A4): A1) Regulate plant stress tolerance; A2) Cultivate plants resistant to reverse gene; A3) Preparation of products for regulating plant stress tolerance; A4) Preparation of products for breeding plants resistant to reverse gene mutation; The protein is any one of the following B1)-B4): B1) The amino acid sequence is the protein shown in SEQ ID NO:2; B2) A fusion protein having the same function obtained by connecting a tag to the N-terminus and/or C-terminus of the amino acid sequence shown in SEQ ID NO: 2; B3) A protein having the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 2; B4) A protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and has the same function. 2. Use of a biological material related to the protein of claim 1 in any of the following A1)-A4): A1) Regulate plant stress tolerance; A2) Cultivate plants resistant to reverse gene; A3) Preparation of products for regulating plant stress tolerance; A4) Preparation of products for breeding plants resistant to reverse gene mutation; The biological material is any one of the following E1) to E5): E1) a nucleic acid molecule encoding the protein according to claim 1; E2) an expression cassette containing the nucleic acid molecule described in E1); E3) a recombinant vector containing the nucleic acid molecule described in E1), or a recombinant vector containing the expression cassette described in E2); E4) a recombinant microorganism containing the nucleic acid molecule described in E1), or a recombinant microorganism containing the expression cassette described in E2), or a recombinant microorganism containing the recombinant vector described in E3); E5) A recombinant host cell containing the nucleic acid molecule of E1), or a recombinant host cell containing the expression cassette of E2), or a recombinant host cell containing the recombinant vector of E3). 3. The use according to claim 2, characterized in that: E1) the nucleic acid molecule is any one of the following: F1) the DNA molecule shown in SEQ ID NO: 21-839; F2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in F1) and encodes the protein. 4. The use according to any one of claims 1 to 3, characterized in that the stress resistance is salt resistance. 5. The use according to any one of claims 1 to 4, characterized in that the plant is a dicotyledonous plant or a monocotyledonous plant. 6. A method for cultivating plants resistant to gene reversal, comprising the following steps: increasing the content and/or activity of the protein of claim 1 in a target plant to obtain a transgenic plant; the transgenic plant has higher stress tolerance than the target plant. 7. The method according to claim 6, characterized in that the stress resistance is salt resistance. 8. The method according to claim 7, characterized in that: the method for increasing the content and/or activity of the protein described in claim 1 in the target plant is to overexpress the protein described in claim 1 in the target plant. 9. The method according to any one of claims 6 to 8, characterized in that: the overexpression method is to introduce the coding gene of the protein described in claim 1 into the target plant. 10. The method according to any one of claims 6 to 9, characterized in that the plant is a dicotyledon or a monocotyledon.