OsWRKY72 protein and its coding gene in regulating plant salt stress resistance

Abstract

The application discloses an OsWRKY72 protein and application of a coding gene thereof in regulation of plant salt stress resistance, relates to the technical field of plant genetic engineering, and the OsWRKY72 protein has any one of the following amino acid sequences: an amino acid sequence shown in SEQ ID NO:2; or an amino acid sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2. The application provides a gene resource for cultivating and improving a new salt-tolerant plant variety, and provides a theoretical basis for clarifying a molecular mechanism of the OsWRKY72 in a plant salt stress adversity signal response.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to the application of an OsWRKY72 protein and its encoding gene in regulating plant salt stress resistance. Background Technology

[0002] Currently, there are approximately 955 million hectares of land in the world. 2 Saline-alkali land accounts for 10% of my country's land area, mainly distributed in the northwest inland and southeast coastal regions. With global climate change and inadequate irrigation methods, the area of ​​saline-alkali soil is increasing year by year, seriously threatening ecological sustainability and food security. At the same time, the global population is constantly increasing, making it imperative to utilize saline-alkali soil for the cultivation of major crops to meet food demand.

[0003] Rice is one of the world's most important food crops, providing food for more than half of the global population. However, rice is also highly sensitive to salt stress, exhibiting varying sensitivities at different stages of its growth and development, and different genotypes of rice also demonstrate differing responses to salt stress. Therefore, in-depth research into the mechanisms regulating salt tolerance in rice, while simultaneously improving its salt tolerance and cultivating new rice varieties with good salt tolerance, is an important way to fully utilize tidal flat resources and increase rice yield.

[0004] Plant transcription factors are widely involved in various biological processes, including plant growth, development, and stress responses. The WRKY gene family is one of the largest transcription factor families in higher plants, playing a crucial role in some processes of plant response to biotic and abiotic stresses. WRKY family genes regulate stress responses by binding to the W-box [TGACC(A / T)] on the promoters of their target genes, thereby activating or repressing the expression of downstream genes. Abiotic stresses can induce the expression of WRKY transcription factors and trigger signaling cascade networks to enhance plant resistance. In rice, the WRKY family has at least 102 members, but the functional characteristics of most members remain unclear.

[0005] Therefore, isolating and cloning this family of genes from rice and identifying its function in improving rice salt tolerance is of great significance for breeding new salt-tolerant rice varieties. Summary of the Invention

[0006] In view of this, the main objective of the present invention is to provide an application of the OsWRKY72 protein and its encoding gene in regulating plant salt stress resistance.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] Application of OsWRKY72 protein or its encoding gene, or biological materials containing its encoding gene in regulating plant salt stress resistance;

[0009] The OsWRKY72 protein has any one of the following amino acid sequences:

[0010] 1) The amino acid sequence shown in SEQ ID NO: 2; or

[0011] 2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2.

[0012] It should be noted that in this invention, amino acid modification is performed through amino acid substitution, deletion, or insertion. Amino acid modification can be introduced into the protein using conventional techniques in the art. Specifically, the substitution includes replacing a certain amino acid with an amino acid having a similar side chain. This similar side chain is defined based on conventional understanding in the art, including having similar basic side chains, such as lysine, arginine, and histidine; similar acidic side chains, such as aspartic acid and glutamic acid; and similar nonpolar or polar side chains. Amino acid substitution can also be based on the hydrophilicity / hydrophobicity of the amino acid, for example, substitution to improve the hydrophilicity / hydrophobicity of the amino acid; or amino acid substitution to improve the stability of the antibody protein sequence. The aforementioned changes in the amino acid sequence will also lead to corresponding changes in the nucleotide sequence expressing the amino acid sequence.

[0013] Furthermore, the gene encoding the OsWRKY72 protein has any one of the following nucleotide sequences:

[0014] 1) The nucleotide sequence shown in SEQ ID NO: 1; or

[0015] 2) The nucleotide sequence encoding a protein with the same function obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO: 1.

[0016] Furthermore, the regulation is a positive regulation.

[0017] Furthermore, the regulation also includes the OsWRKY72 protein regulating the expression of the OsWRKY72 gene by binding to the W-box element in the promoter of the OsWRKY72 gene.

[0018] Furthermore, the promoter has 5 W-box elements.

[0019] Furthermore, the plant is a monocotyledonous plant.

[0020] Furthermore, the monocotyledonous plant is one of rice, corn, or wheat.

[0021] Furthermore, the biomaterial is a recombinant vector, engineered bacteria, or host cell.

[0022] The present invention also provides a method for improving the salt tolerance of plants, including increasing the expression level of OsWRKY72 protein in plants, wherein the amino acid sequence of OsWRKY72 protein is shown in SEQ ID NO: 2.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] This invention cloned the rice OsWRKY72 gene from the highly adaptable indica rice variety Minghui 86. Using MH86 as background material, two homozygous mutant lines, wrky72-1 and wrky72-2, were obtained through CRISPR / Cas9 technology and Agrobacterium-mediated transformation. Simultaneously, two overexpressing transgenic lines, WRKY72-OE1 and WRKY72-OE2, were obtained through Agrobacterium-mediated transformation using the strong promoter Ubi to drive the WRKY72 coding sequence. Phenotypic analysis revealed that the WRKY72 overexpressing lines exhibited stronger salt tolerance, while the wrky72 mutant was more sensitive to salt stress. These results indicate that WRKY72 can coordinate the rice response to salt stress and has significant application value in enhancing rice salt tolerance. This provides gene resources for breeding and improving new salt-tolerant plant varieties and provides a theoretical basis for elucidating the molecular mechanism of OsWRKY72 in plant salt stress stress signal response. Attached Figure Description

[0025] Figure 1 This is an agarose gel electrophoresis image of the WRKY72 CDS amplification products;

[0026] Figure 2 This is a schematic diagram of the WRKY72 mutation types and proteins in MH86, where A: schematic diagram of the WRKY72 gene structure; B: type diagram of the two mutants wrky72-1 and wrky72-2; C: schematic diagram of the proteins of the two mutants wrky72-1 and wrky72-2.

[0027] Figure 3 The relative expression levels of WRKY72 in the WT strain, overexpression strains WRKY72-OE1 and WRKY72-OE2, and mutant strains wrky72-1 and wrky72-2;

[0028] Figure 4 This is a graph showing the salt tolerance results of the WT strain, the overexpression strains WRKY72-OE1 and WRKY72-OE2, and the mutant strains wrky72-1 and wrky72-2. In the graph, A: Salt stress treatment results; B: Survival rate bar graph.

[0029] Figure 5 The results show the results of different gene regulation experiments on the WRKY72 gene; (a) the relative expression level of WRKY72; (b) the Y1H experiment results; (c) the EMSA experiment results; and (d) the Luciferase experiment results. Detailed Implementation

[0030] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0032] Example 1: Cloning of the rice WRKY72 gene

[0033] After searching the NCBI website, corresponding primers were designed, and the WRKY72 gene was cloned using indica rice MH86 cDNA as a template. The amplification primers are as follows:

[0034] WRKY72-CDS-F:ATGGAGAACTTCCCGATACTCTTTG

[0035] WRKY72-CDS-R:CTACTGGAACATGTGGGAAGCA

[0036] Amplification was performed using the PCR amplification enzyme KOD FX DNA Polymerase provided by TOYOBO. The PCR amplification system consisted of: Primer Star 10 μL, ddH2O 7 μL, Primer-F 1.5 μL, Primer-R 1.5 μL, and template DNA 1 μL. After adding the sample, the mixture was mixed and centrifuged. The PCR amplification program was as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 s, 65℃ annealing for 30 s, 72℃ extension for 60 s, for a total of 35 cycles; 72℃ extension for 5 min; and isothermal at 4℃. This yielded the WRKY72 gene CDS from MH86. The PCR products were detected by agarose gel electrophoresis after amplification. The agarose gel electrophoresis image is shown below. Figure 1 As shown, the amplification product was recovered via gel electrophoresis and sent to the company for sequencing to obtain the WRKY72 gene CDS, the specific sequence of which is as follows (SEQ ID NO: 1):

[0037] ATGGAGAACTTCCCGATACTCTTTGCGACGCAGCCGACGTCGTCTTCTACCTCCAGCTCCTACCACTTCATGAGCTCCTCTTCCGGCAGCCATGACCACCGCCACCACCATGGACTACAGGCAGGCGGCAACGGCGGTGGCGGTGGTGGAAGCTTGAGCCATGGCTTGTTCATGGGGTCGTCGTCGTCGTCAATTAGGATGGAGGAGCTCTCCAACTCCAAGCAGGCCGGCGACGTCGTCGTCGACGGTGGTGCAACGAGGAGCCCACACGGCGGCGACGGAGACGGAGCTGCGGGCGACGACGGCGGCGATGCGCAGGCCGCCGCCGCCGGAGGGAGGAAGAAGGGGGAGAAGAAGGAGCGGCGGCCGCGGTTCGCGTTCCAGACGCGCAGCCAGGTCGACATCCTCGACGACGGCTACCGGTGGAGGAAGTACGGCCAGAAGGCCGTCAAGAACAACAAATTCCCAAGGAGCTACTACCGGTGCACGCACCAAGGGTGCAACGTGAAGAAGCAGGTGCAGCGGCTGTCGCGGGACGAGACGGTGGTGGTGACGACGTACGAGGGCACGCACACGCACCCCATCGAGAAATCCAACGACAACTTCGAGCACATCCTCACCCAAATGCACATCTACTCCGGCCTCACGCCTTCATCGGCTGCGCATGCCTCATCCTCCTCACCTCTCTTCCCTTCAGCTGCTGCTGCTGCTTCCCACATGTTCCAGTAG;

[0038] The protein sequence of WRKY72 is as follows (SEQ ID NO: 2):

[0039] MENFPILFATQPTSSSTSSSYHFMSSSSGSHDHRHHHGLQAGGNGGGGGGSLSHGLFMGSSSSIRMEELSNSKQAGDVVVDGGATRSPHGGDGDGAAGDDGGDAQAAAAGGRKKGEKKER RPRFAFQTRSQVDILDDGYRWRKYGQKAVKNNKFPRSYYRCTHQGCNVKKQVQRLSRDETVVVTTYEGTHTHPIEKSNDNFEHILTQMHIYSGLTPSSAAHASSSSPLFPSAAAAASHMFQ.

[0040] Example 2: Creation of WRKY72 mutant lines and overexpression lines

[0041] The WRKY72 gene accession number LOC_Os11g29870 was entered into the CRISPR-GE website (http: / / skl.scau.edu.cn / home / ) for CRISPR / Cas9 knockout target prediction. Simultaneously, suitable target sequences were selected as gRNA targets from the early coding region and conserved protein kinase regions, such as... Figure 2 As shown in Figure A, the sequence includes Target1 and Target2. Target1's sequence is shown in SEQ ID NO: 3, specifically: GGTGGTGGAAGCTTGAGCCA; Target2's sequence is shown in SEQ ID NO: 4, specifically: TGGGAATTTGTTGT TCTTGA. The target sequences were then entered into the CRISPR RGEN Tools website (http: / / www.rgenome.net / cas-offinder / ) for off-target analysis. The following targets were then selected:

[0042] WRKY72-Cas9-T1 (SEQ ID NO: 5):AATAATGGTCTCTGGCGGTGGTGGAAGCTTGAGCCAGTTTTAGAGCTAGAAATAGC

[0043] WRKY72-Cas9-T2 (SEQ ID NO: 6):ATTATTGGTCTCTAAACTGGGAATTTGTTGTTCTTGAGCTTCTTGGTGCC;

[0044] Then, PCR amplification was performed to introduce the target site. Finally, a CRISPR / Cas9 knockout vector for the pHUE411 gene was constructed using the Golden Gate cloning method. After correct sequencing, the recombinant plasmid was transformed into Agrobacterium (EHA105) competent cells using a freeze-thaw method. Using MH86 as background material, transgenic rice was obtained through Agrobacterium-mediated genetic transformation. DNA was extracted from the obtained T0 generation transgenic plants, and CRISPR / Cas9 vector-specific primers were used to detect whether they were positive plants. The primer sequences are as follows:

[0045] OsU3-FD3 (SEQ ID NO: 7): 5'-GACAGGCGTCTTCTACTGGTGCTAC-3'

[0046] TaU3-RD (SEQ ID NO: 8): 5'-CTCACAAATTATCAGCACGCTAGTC-3';

[0047] Positive plants were planted in the experimental field. After harvesting individual T1 generation seedlings and extracting DNA from their leaves, primers were designed near the two knockout target sites to detect the knockout effect. The primer sequences are as follows:

[0048] WRKY72-Cas9-T1T2-F (SEQ ID NO: 9):CCCTGGTTGTAACTTGTGAAACT

[0049] WRKY72-Cas9-T1T2-R (SEQ ID NO: 10):TCCCAAAATCAAACATCAAAGAC;

[0050] It should be noted that the T0 generation transgene detection can preliminarily determine whether the transgene is positive using OsU3-FD3 and TaU3-RD primers, because these primers are knockout vector-specific primers. The specific knockout mutation needs to be detected using primers designed for the WRKY72 gene genome (the mutation type is determined by the WRKY72-Cas9-T1T2-F and WRKY72-Cas9-T1T2-R primers).

[0051] Two homozygous mutant lines, wrky72-1 and wrky72-2, were obtained through testing. Figure 2 B), and it was also found that the mutations caused changes in protein translation in both mutants (e.g. Figure 2 C).

[0052] Two overexpressing transgenic lines, WRKY72-OE1 and WRKY72-OE2, were obtained by Agrobacterium-mediated genetic transformation of rice with the strong promoter Ubi driving the WRKY72 coding sequence.

[0053] Example 3: Gene expression levels of the WRKY72 gene in different rice lines

[0054] wrky72-1, wrky72-2, WRKY72-OE1, WRKY72-OE2, and WT (Indica rice MH86) were planted in experimental fields. After normal cultivation under natural conditions, RNA was extracted from leaves of the corresponding lines and converted into cDNA. The expression levels were detected using quantitative real-time fluorescence technology. It was found that the expression level of WRKY72 in the overexpression lines (WRKY72-OE1 and WRKY72-OE2) was significantly higher than that in the wild type and the wrky72 mutants (wrky72-1 and wrky72-2). Specific results are shown in Table 1 below. Figure 3 As shown.

[0055] The fluorescent primer sequences are as follows:

[0056] WRKY72-qPCR-F (SEQ ID NO: 11):GTCGTCTTCTACCTCCAG

[0057] WRKY72-qPCR-R (SEQ ID NO: 12): GCTCCTCCATCCTAATTGA

[0058] Table 1. Relative expression levels of WRKY72 gene in different strains.

[0059]

[0060] Example 4: Determination of salt tolerance of different strains

[0061] Seeds of wrky72-1, wrky72-2, WRKY72-OE1, and WRKY72-OE2 were planted in an experimental field. After four weeks of normal cultivation, rice seedlings were subjected to salt stress treatment with 200 mM NaCl for 7 days, followed by rehydration for 5 days, and the survival rate was recorded. The results showed that compared with the wild type, the WRKY72 mutants (wrky72-1 and wrky72-2) were more sensitive to salt stress, while the overexpression lines (WRKY72-OE1 and WRKY72-OE2) were more salt-tolerant (e.g., ...). Figure 4 A). Five days after rehydration, the survival rate of the WRKY72-expressing lines was found to be significantly higher than that of the wrky72 mutant and wild type. Specific results are shown in Table 2 below. Figure 4 As shown in B.

[0062] Table 2 Survival rates of different strains after salt treatment

[0063] 50.04 16.68 8.34 62.55 66.72 54.21 20.85 16.68 66.72 66.72 50.04 12.51 16.68 58.38 58.38 45.87 16.68 12.51 54.21 58.38

[0064] Example 5

[0065] To further clarify the mechanism of WRKY72 in regulating salt stress, this embodiment analyzes the WRKY72 promoter and finds that the WRKY72 promoter has 5 W-box elements, including W1:TTGACT; W2:TTGACC; W3:TTGACT; W4:TTGACC; W5:TTGACT;

[0066] Using quantitative real-time primers designed for the WRKY72 gene 5-UTR, it was found that the transcriptional level of WRKY72 was significantly increased in its overexpression lines, as shown in the following results. Figure 5 As shown in a.

[0067] The primer sequences for quantitative fluorescence are as follows:

[0068] WRKY72-5UTR-qPCR-F (SEQ ID NO: 13):ATTAAGTAGAGGGTTTATTTAGC;

[0069] WRKY72-5UTR-qPCR-R (SEQ ID NO: 14):GAATAGCTCAGTTGGATAGA;

[0070] The following experiments using yeast monohybrid (Y1H), EMSA, and Luciferases were used to verify whether WRKY72 can regulate its own expression.

[0071] 1. Y1H experimental procedure:

[0072] Transcription factors were constructed into the pB42AD vector, and a promoter containing a potential W-BOX binding element was constructed into the Placzi vector. Yeast transformation was performed using the EGY48 yeast strain. After transformation, the yeast samples were plated onto Trp-Ura bicellular plates and incubated upside down at 28°C or 30°C. Two days later, the experimental and control yeast samples were plotted onto X-gal chromogenic plates and incubated at 28°C or 30°C. Interactions were observed after one or two days. Results are as follows: Figure 5 As shown in b.

[0073] 2. EMSA Experiment

[0074] (1) First, based on the type of W-BOX element that the transcription factor may bind, the main W-BOX element to be bound is located on the promoter. A probe is designed using 26 bp of the WRKY72 promoter (containing the w-box). The probe is labeled with cy5.5, and an unlabeled sequence is used as a competing cold probe. The probe sequence is as follows:

[0075] W1-cy5.5: CAATTTCGATAGTCAAAGTGAAAAAA (SEQ ID NO: 15);

[0076] W4-cy5.5: ATATTTTGCTTTGACCTGGTTGAAAA (SEQ ID NO: 16);

[0077] W5-cy5.5: GTGCCTTGCCAGTCAACCTTAAATAT (SEQ ID NO: 17);

[0078] (3) Transcription factors were constructed into the pET-32a prokaryotic expression vector to induce the His-WRKY72 fusion protein, which was expressed in BL21(DE3) and then expressed using BeyoGold. TM His-tag purification resin (Beyotime Biotechnology, China) for protein purification;

[0079] (4) Prepare 4% or 6% polyacrylamide gel, mix the protein and probe and add it to the sample reaction at 25°C for 40 min, add the reaction product to the sample well and electrophoresis at 80V for 45 min.

[0080] (5) Take a picture at a wavelength of 700 nm after electrophoresis;

[0081] The results are as follows Figure 5 As shown in c.

[0082] 3. Luciferases experiment

[0083] The full-length coding sequence of WKRY72 was constructed into the pRTVcGFP vector with BamHI and HindIII restriction sites, generating an effector (Ubipro:WRKY72:GFP). The WRKY72 promoter (2kb upstream of ATG) was amplified and constructed into the PgreenII0800-LUC vector with HindIII and BamHI restriction sites as a reporter gene. After plasmid extraction, it was co-transformed into rice protoplasts and cultured at 28℃ in the dark for 12-16 hours. The activities of the reporter gene Firefly luciferase (LUC) and Renilla substrate (REN) were detected using a microplate reader using the Dual Luciferase Reporter Assay Kit from Novizumab. The emission wavelength of Luciferin catalyzed by Firefly luciferase was 560nm, and the emission wavelength of Coelen-terazine catalyzed by Renilla luciferase was 465nm. The LUC / REN ratio was used for analysis. Each plasmid combination underwent five independent transformations, and the experiment was repeated three times. The results are as follows: Figure 5 As shown in d.

[0084] Experiments using yeast monohybrid (Y1H), EMSA, and Luciferases showed that WRKY72 can bind to its own promoter region W-box site to positively regulate autologous expression.

[0085] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0086] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Application of overexpressing the OsWRKY72 protein-coding gene or biological materials containing overexpressing the OsWRKY72 protein-coding gene in improving rice salt stress resistance; The OsWRKY72 protein has the amino acid sequence shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The gene encoding the OsWRKY72 protein has the nucleotide sequence shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The promoter contains 5 W-box elements.

4. The application according to claim 1, characterized in that, The biomaterial is a recombinant vector, engineered bacteria, or host cell.

5. A method for improving the salt tolerance of rice, characterized in that, This includes overexpressing the OsWRKY72 protein encoding gene to increase the expression level of OsWRKY72 protein in rice, the amino acid sequence of which is shown in SEQ ID NO: 2.

Images