Application of transcription factor TabHLH168 protein in wheat disease resistance

Abstract

The invention relates to the technical field of gene engineering, and discloses application of transcription factor TabHLH168 protein in wheat disease resistance. The amino acid sequence of the protein TabHLH168 is as shown in SEQ ID NO. 1; 1, and the coding gene sequence of the TabHLH168 protein is as shown in SEQ ID NO. 2. And the protein TabHLH168 is a target protein of the Fusarium pseudograminearum effect protein FpE02. According to the application of the transcription factor TabHLH168 protein in wheat disease resistance, the coding gene of the TabHLH168 protein is overexpressed, transgenic wheat which can be inherited stably and has wheat stem rot resistance is obtained, a new solution is provided for wheat disease resistance breeding, and the application has important significance in the field of disease-resistant wheat breeding.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to the application of transcription factor TabHLH168 protein in wheat disease resistance. Background Art

[0002] Wheat stem rot, caused by Fusarium pseudograminearum, is an important disease in wheat production. When it occurs seriously, it can cause a 35% reduction in wheat yield. This disease was first reported in my country. Due to the lack of knowledge about the pathogenic mechanism of the pathogen, there is a lack of targeted and effective prevention and control measures and technologies in production, resulting in the rapid spread of the disease in my country's winter wheat areas and serious damage, which has seriously endangered wheat production. The application of resistant varieties is the most economical and effective measure to prevent and control diseases, but most of the main cultivated varieties in production are highly susceptible or susceptible varieties, and there is a lack of resistant varieties to this disease. Therefore, in-depth research on the molecular mechanism of wheat disease resistance, the discovery of stem rot resistance genes and the cultivation of stable disease-resistant varieties are the main tasks of wheat breeding for stem rot resistance at this stage. While enhancing wheat disease resistance, wheat yield must also be taken into account to meet wheat supply needs. Summary of the invention

[0003] The purpose of the present invention is to provide an application of the transcription factor TabHLH168 protein in wheat disease resistance, and to overexpress the coding gene of the TabHLH168 protein to obtain transgenic wheat with wheat stem rot resistance that can be stably inherited, thereby providing a new solution for carrying out wheat disease resistance breeding, and having important significance in the field of disease-resistant wheat breeding.

[0004] To achieve the above object, the present invention provides the use of transcription factor TabHLH168 protein in wheat disease resistance, the amino acid sequence of TabHLH168 protein is shown in SEQ ID NO.1; the coding gene sequence of TabHLH168 protein is shown in SEQ ID NO.2.

[0005] Furthermore, TabHLH168 protein is the target protein of the F. graminearum effector protein FpE02.

[0006] Furthermore, overexpression of TabHLH168 protein in wheat plants improves the resistance of wheat plants to stem base rot.

[0007] The present invention also provides a recombinant vector, which comprises the gene sequence shown as SEQ ID NO.2.

[0008] Furthermore, the recombinant vector is a plant overexpression vector.

[0009] The present invention also provides application of the above-mentioned recombinant vector in cultivating wheat resistant to stem base rot.

[0010] The present invention also provides a method for cultivating a wheat variety resistant to stem base rot, which comprises constructing the above-mentioned recombinant vector, and then introducing the recombinant vector into wheat plants to obtain a new transgenic wheat variety.

[0011] The advantages and positive effects of the application of the transcription factor TabHLH168 protein in wheat disease resistance are:

[0012] 1. The present invention uses forward genetics methods to confirm that overexpression of the transcription factor protein TabHLH168 can enhance wheat's resistance to stem rot. During the identification of wheat stem rot resistance, the resistance of the transgenic strains was further improved compared to normal wheat plants, proving the disease resistance function of the transcription factor protein TabHLH168.

[0013] 2. The present invention establishes a method for cultivating wheat-resistant varieties from the dimensions of molecular biology and genetic engineering, and proves the feasibility of the method through results, thereby providing a new solution for carrying out disease-resistant wheat breeding, and has a significant contribution in the field of disease-resistant wheat breeding.

[0014] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is to verify the interaction between the transcription factor protein TabHLH168 and the effector protein FpE02 by immunoprecipitation in Example 1 of the present invention;

[0016] Figure 2 The expression profile of the TabHLH168 gene in the interaction between wheat and Fusarium graminearum in Example 2 of the present invention;

[0017] Figure 3 is the positive verification result of the transgenic wheat in Example 3 of the present invention, wherein a is the electrophoresis verification result of the overexpression strain; b is the relative expression level of the TabHLH168 gene in the transgenic strains H168-L3 and H168-L6;

[0018] Figure 4 The results of disease resistance identification of transgenic wheat in Example 3 of the present invention are shown, wherein a represents the disease susceptibility of the stem base of transgenic wheat and wild type wheat, and b represents the disease index of each treatment. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0020] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0021] Unless otherwise defined, the instruments, equipment and reagents used in the present invention are conventionally commercially available.

[0022] Source of wild-type strain 2035 of Pseudomonas graminearum: isolated and preserved from diseased tissue of wheat stem base at Wuqiao by Fungal Disease Laboratory, Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences.

[0023] Source of Shixin 828 wheat seeds: bred and donated by Zhaoxian Experimental Base of Shijiazhuang Academy of Agriculture and Forestry Sciences.

[0024] The present application adopts the immunoprecipitation method to screen out the target protein TabHLH168 of the pathogenicity-related effector protein FpE02 of Pseudomonas graminearum. The protein TabHLH168 is a basic helix-loop-helix transcription factor. The amino acid sequence and gene coding (CDS) sequence of the transcription factor TabHLH168 are obtained from the NCBI website. Using the Agrobacterium-mediated method, the target gene sequence is introduced into wheat to obtain a transformant strain that can be stably inherited; then the relative expression level of the transcript of the transgenic strain and the resistance of the transgenic strain to wheat stem base rot are detected to verify whether the resistant variety has been successfully cultivated. The following is a specific description through examples.

[0025] Example 1 Identification and bioinformatics analysis of transcription factor TabHLH168 protein

[0026] The target protein in plants of the pathogenicity-related F. graminearum effector protein FpE02 obtained by screening was identified: the target protein that interacts with the effector protein FpE02 was obtained through immunoprecipitation experiments. It was identified that the target protein is the transcription factor protein TabHLH168 ( Figure 1 shown).

[0027] The sequence information of the transcription factor protein TabHLH168 was obtained from the NCBI website, with the index number XP_044352556. The amino acid sequence of the transcription factor protein TabHLH168 is shown in SEQ ID NO.1, the amino acids at positions 29-30, 32, 37, and 74-75 are DNA binding sites, and the amino acids at positions 22-100 are basic helix-loop-helix domains. The CDS sequence length of the gene encoding the protein is 660 bp.

[0028] Example 2 Expression profile of TabHLH168 gene in the interaction between wheat and Fusarium graminearum

[0029] This example provides RT-PCR detection of the expression of TabHLH168 gene induced by F. graminearum.

[0030] 1. Preparation of test materials:

[0031] 1) The wild-type strain 2035 of Pseudomonas graminearum was activated and cultured on PDA solid medium. Three pieces of Pseudomonas graminearum cakes at the edge of the colony were taken using a 5 mm borer and placed in 100 mL of sterilized carboxylmethyl cellulose (CMC) liquid medium. After shaking culture at 25 ° C and 180 rpm for 4 to 5 days, a spore suspension was prepared after filtering through gauze. The concentration was 1×10 5 spores / mL for later use.

[0032] 2) Selection and treatment of wheat varieties: Select Shixin 828 wheat seeds with full grains, disinfect them with 1% sodium hypochlorite for 5 minutes, wash them three times with sterile water, immerse them in the prepared conidia suspension for 5 minutes, and place them with tweezers in a culture dish with a diameter of 9 cm and covered with three layers of filter paper, with 10 seeds in each dish. Culture them in a constant temperature and light incubator at 22°C for 4 days, inoculate the spore suspension at the base of the wheat stem, 20 μL per plant, and take tissue samples from the base of the wheat stem at 0h, 12h, 24h, 3d, 5d, and 7d. The samples were quickly stored in a -80°C refrigerator for total RNA extraction.

[0033] 2. qRT-PCR detection of relative expression of TabHLH168 gene:

[0034] RNA extraction and first-strand cDNA synthesis: RNA extraction, total RNA was extracted using the RNAprep Pure kit (Tiangen Company), and the extracted RNA was purified. Then the first strand was synthesized using the RevertAid TM first-strand cDNA synthesis kit to obtain cDNA from each sampling point. The operation steps are detailed in the instructions. The wheat elongation factor TaEF-1α gene (GenBank accession number: M90077.2) was used as the internal reference gene, and primers were designed based on the TabHLH168 gene sequence for qRT-PCR analysis.

[0035] Primers used for qRT-PCR analysis were designed as follows:

[0036] TabHLH168Q-F: 5'-CCGTTGGCTGCTCATCCTC-3' (SEQ ID NO.3);

[0037] TabHLH168Q-R: 5'-CGCTGCCGTCTGTGACTTGTAG-3' (SEQ ID NO.4);

[0038] TaEF-1α-F: 5'-TGGTGTCATCAAGCCTGGTATGGT-3' (SEQ ID NO.5);

[0039] TaEF-1α-R: 5'-ACTCATGGTGCATCTCAACGGACT-3' (SEQ ID NO. 6).

[0040] Before using the primers, the specificity and amplification efficiency of the amplified products should be tested (≥90%). Premix Ex Taq TM (Takara, Beijing, China) and ABIQuantStudio 6 Flex quantitative PCR instrument (ThermoFisher, America), according to the instructions, qRT-PCR was performed with cDNA from each sampling point as template. Each reaction was repeated three times, and the Ct value was averaged. The Delta Delta Ct method was used to analyze the experimental data. The Ct value of the 0h sampling point was used as a control, and the value was set to 1 to determine the relative expression of the TabHLH168 gene at each sampling point.

[0041] The results of qRT-PCR are as follows Figure 2 As shown, after inoculation with F. graminearum, the expression of TabHLH168 gene was downregulated during the infection period, indicating that F. graminearum inhibited the expression of TabHLH168 gene.

[0042] Example 3 Verification of resistance to overexpression of transcription factor protein encoding gene TabHLH168

[0043] 1. Preparation of wheat transformant strains:

[0044] The target gene was obtained according to the results of Example 1. Full-length primers carrying the recombinant fragment were designed to amplify the full-length fragment of the TabHLH168 gene, and connected to the pOE3 plant expression vector by recombination to obtain the overexpression vector TabHLH168-OE3.

[0045] The full-length primer sequence carrying the recombinant fragment is as follows:

[0046] TabHLH168O-F:5'-GCCATGGAGGCCAGTGAATTCATGAAGAGCAGGA GGCAGAG-3' (SEQ IDNO.7);

[0047] TabHLH168O-R: 5'-CAGCTCGAGCTCGATGGATCCTCATGTCATGAGG ACGGAGG-3' (SEQ ID NO. 8).

[0048] Through Agrobacterium-mediated transfection in the common spring wheat variety JW1, a stable overexpression transformant strain was obtained, which can overexpress the TabHLH168 gene. The genomic DNA of the T3 transgenic wheat and the control wheat (JW1) was extracted by the CTAB method, and the inserted fragment was detected and sequenced using the detection primer pOE-F / R:

[0049] pOE-F:5'-GCCCTGCCTCATACGCT-3' (SEQ ID NO.9);

[0050] pOE-R: 5'-GCTCACCCTGTTGTTTGG-3' (SEQ ID NO. 10).

[0051] After the experiment, two transformant strains capable of stable inheritance were obtained, which were respectively recorded as H168-L3 and H168-L6. The relative expression level of the transcript of the TabHLH168 gene in the transformant strains was detected according to the method of Example 2. Figure 3 As shown, the TabHLH168 gene was significantly overexpressed in both H168-L3 and H168-L6 lines compared with wild-type wheat (JW1).

[0052] 2. Analysis of disease resistance of transgenic wheat:

[0053] A spore suspension of Fusarium graminearum was prepared according to the method of Example 2. The two transgenic strains H168-L3 and H168-L6 and the wild-type JW1 variety were disinfected with 1% sodium hypochlorite for 5 minutes, washed three times with sterile water, immersed in the prepared conidia suspension for 5 minutes, and placed with tweezers in a culture dish with a diameter of 9 cm and covered with three layers of filter paper, with 10 seeds in each dish. The seeds were cultured in a constant temperature and light incubator at 22°C for 4 days. The spore suspension was inoculated at the base of the wheat stem, 20 μL per plant, and the disease was investigated after culturing in a moisturizing box for 5 days.

[0054] Grading Standard:

[0055] Level 0: no browning symptoms;

[0056] Level 1: The length of browning below the first leaf sheath accounts for less than 25% of the stem;

[0057] Level 2: The length of browning below the first leaf sheath accounts for 25-50% of the stem;

[0058] Level 3: The length of browning below the first leaf sheath accounts for 50-75% of the stem, and the brown color deepens;

[0059] Level 4: The brown length below the first leaf sheath accounts for more than 75% of the stem, or the entire plant turns brown and dies.

[0060] Disease index = ∑ (number of diseased plants × representative values ​​of each level) / (total number of plants surveyed × highest representative value) × 100%.

[0061] Figure 4 The disease susceptibility of wheat stem base tissues in each treatment showed that the wheat stem base rot in the control group had spread to the entire leaf sheath, while the disease expansion of the two transgenic lines H168-L3 and H168-L6 was significantly weakened. The statistical results of the disease index showed that compared with the control group, the disease index of the transgenic lines H168-L3 and H168-L6 was significantly (P<0.01) reduced.

[0062] Therefore, the present invention adopts the application of the above-mentioned transcription factor TabHLH168 protein in wheat disease resistance, overexpresses the coding gene of the TabHLH168 protein, and obtains transgenic wheat with wheat stem rot resistance that can be stably inherited, which provides a new solution for carrying out wheat disease resistance breeding and is of great significance in the field of disease-resistant wheat breeding.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of transcription factor TabHLH168 protein in wheat disease resistance, characterized in that: The amino acid sequence of the TabHLH168 protein is shown in SEQ ID NO.1; the coding gene sequence of the TabHLH168 protein is shown in SEQ ID NO.

2.

2. The use of the transcription factor TabHLH168 protein in wheat disease resistance according to claim 1, characterized in that: TabHLH168 protein is the target protein of the F. graminearum effector protein FpE02.

3. The use of the transcription factor TabHLH168 protein in wheat disease resistance according to claim 1, characterized in that: Overexpression of TabHLH168 protein in wheat plants improves the resistance of wheat plants to stem base rot.

4. A recombinant vector, characterized in that: The recombinant vector comprises the gene sequence shown as SEQ ID NO.

2.

5. The recombinant vector according to claim 4, characterized in that: The recombinant vector is a plant overexpression vector.

6. Use of the recombinant vector as claimed in claim 5 in cultivating wheat resistant to stem base rot.

7. A method for breeding wheat varieties resistant to stem rot, characterized in that: The recombinant vector as claimed in claim 4 is constructed, and then the recombinant vector is introduced into wheat plants to obtain a new transgenic wheat variety.

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