Application of transcription factor tabhlh168 protein in wheat disease resistance

Abstract

The application relates to the technical field of genetic engineering, and discloses application of a transcription factor TabHLH168 protein in wheat disease resistance. The amino acid sequence of the TabHLH168 protein is shown as SEQ ID NO. 1; the coding gene sequence of the TabHLH168 protein is shown as SEQ ID NO. 2. The TabHLH168 protein is a target protein of a false smut fungus effect protein FpE02. The application uses the above-mentioned application of the transcription factor TabHLH168 protein in wheat disease resistance, overexpresses the coding gene of the TabHLH168 protein, obtains transgenic wheat with stable heredity and wheat stem base rot resistance, provides a new solution for carrying out wheat disease resistance breeding work, and has important significance in the field of wheat breeding with disease resistance.

Description

TECHNICAL FIELD

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

[0002] Wheat stem base rot caused by Fusarium pseudograminearum is an important disease in wheat production, and can cause 35% reduction in wheat yield. The disease is reported for the first time in China, and due to the little knowledge about the pathogenic mechanism of the pathogen, there is a lack of effective prevention and control measures and technologies, so that the disease has rapidly expanded and caused serious damage in the winter wheat area of China, and has seriously threatened the wheat production. The application of resistant varieties is the most economical and effective measure for disease control, but at present, most of the main cultivated varieties are susceptible or susceptible varieties, and there is a lack of resistant varieties against the disease. Therefore, in-depth study on the molecular mechanism of wheat disease resistance, excavation of stem base rot disease resistance genes and cultivation of stable disease-resistant varieties are the main tasks of wheat breeding against stem base rot at the present stage, and while enhancing the disease resistance of wheat, the yield of wheat should also be considered to meet the demand of wheat supply. SUMMARY

[0003] The application aims to provide application of the transcription factor TabHLH168 protein in wheat disease resistance, overexpression of the coding gene of the TabHLH168 protein, and obtain transgenic wheat with stable heredity and wheat stem base rot resistance, so as to provide a new solution for wheat breeding against disease, and has important significance in the field of disease-resistant wheat breeding.

[0004] In order to achieve the above-mentioned purpose, the application provides application of the transcription factor TabHLH168 protein in wheat disease resistance, the amino acid sequence of the TabHLH168 protein is shown as SEQ ID NO. 1, and the coding gene sequence of the TabHLH168 protein is shown as SEQ ID NO. 2.

[0005] Further, the TabHLH168 protein is a target protein of the Fusarium pseudograminearum effector protein FpE02.

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

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

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

[0009] The application also provides the use of the recombinant vector in cultivating wheat resistant to stem base rot.

[0010] The application also provides a method for cultivating wheat varieties resistant to stem base rot, constructing the recombinant vector, and then introducing the recombinant vector into a wheat plant to obtain a new transgenic wheat variety.

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

[0012] 1. The application uses forward genetics to prove that overexpression of the transcription factor protein TabHLH168 can enhance the disease resistance of wheat to stem base rot. In the wheat stem base rot resistance identification process, the resistance of the transgenic strain is further improved compared with normal wheat plants, proving the disease resistance function of the transcription factor protein TabHLH168.

[0013] 2. The application establishes a method for cultivating wheat resistant varieties from the perspective of molecular biology and genetic engineering, and proves the feasibility of the method, thereby providing a new solution for wheat disease resistance breeding, and having a contribution to the field of disease-resistant wheat breeding.

[0014] The technical solutions of the application will be further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The application example 1 is used to verify the immunoprecipitation of the transcription factor protein TabHLH168 and the effector protein FpE02.

[0016] Figure 2 The application example 2 is used to verify the expression profile of the TabHLH168 gene in the interaction between wheat and Pseudocercosporella herpotrichoides.

[0017] Figure 3 The application example 3 is used to verify the positive results of the transgenic wheat, wherein a is the electrophoresis verification result of the overexpression strain; b is the relative expression amount of the TabHLH168 gene in the transgenic strains H168-L3 and H168-L6.

[0018] Figure 4 The application example 3 is used to verify the disease resistance identification results of the transgenic wheat, wherein a is the disease condition of the stem base of the transgenic wheat and the wild type; b is the disease index of each treatment. DETAILED DESCRIPTION

[0019] The technical solutions of the application will be further described in detail below with reference to the drawings and examples.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

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

[0022] Wild-type strain 2035 of Fusarium oxysporum was isolated and preserved from diseased tissue at the base of wheat stems in Wuqiao, Hebei Academy of Agricultural and Forestry Sciences, Institute of Plant Protection Laboratory.

[0023] The seeds of Shixin 828 wheat originated from the experimental base of Zhaoxian County, Shijiazhuang Academy of Agricultural and Forestry Sciences, and were donated by the family.

[0024] This application employs an immunoprecipitation method to screen for TabHLH168, a target protein of the pathogenicity-related effector protein FpE02 of *Fusarium graminearum*. TabHLH168 is a basic helical-loop-helical transcription factor. The amino acid sequence and code sequence (CDS) of transcription factor TabHLH168 were obtained from the NCBI website. Using Agrobacterium-mediated transformation, the target gene sequence was introduced into wheat to obtain stably heritable transformant lines. The success of resistant variety development was verified by detecting the relative expression levels of transcripts in the transgenic lines and their resistance to wheat stem rot. The following examples illustrate this process.

[0025] Example 1: Identification and Bioinformatics Analysis of Transcription Factor TabHLH168 Protein

[0026] The target protein of the pathogenic Fusarium pseudomonetella effector protein FpE02, obtained through screening, was identified in plants: An immunoprecipitation assay was used to obtain the target protein interacting with FpE02. This target protein was identified as the transcription factor protein TabHLH168. Figure 1 (As shown).

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

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

[0029] This embodiment provides RT-PCR detection of the expression of the TabHLH168 gene induced by Fusarium pseudobulbarum.

[0030] 1. Preparation of experimental materials:

[0031] 1) Wild-type strain 2035 of *Fusarium graminearum* was activated and cultured on PDA solid medium. Three *Fusarium graminearum* mycelial discs from the colony edge were taken using a 5mm punch and placed in 100mL of sterilized carboxymethyl cellulose (CMC) liquid medium. After shaking and culturing at 25℃ and 180rpm for 4–5 days, the culture was filtered through gauze to prepare a spore suspension with a concentration of 1×10⁻⁶. 5 One spore / mL for use.

[0032] 2) Selection and treatment of wheat varieties: Select plump Shixin 828 wheat seeds, disinfect with 1% sodium hypochlorite for 5 min, wash 3 times with sterile water, immerse in the prepared conidial suspension for 5 min, and place 10 seeds in each 9 cm diameter petri dish lined with three layers of filter paper using tweezers. Incubate at 22℃ in a constant temperature and light incubator for 4 days. Inoculate the base of wheat stems with 20 μL of the conidial suspension per plant. Collect tissue samples from the base of wheat stems at 0 h, 12 h, 24 h, 3 d, 5 d, and 7 d. Quickly store the samples in a -80℃ freezer for total RNA extraction.

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

[0034] RNA extraction and first-strand cDNA synthesis: RNA extraction was performed using the RNAprep Pure kit (Tiangen Biotech), and the extracted RNA was purified. First-strand cDNA synthesis was then performed using the RevertAid™ first-strand cDNA synthesis kit to obtain cDNA from each sampling site; detailed operating procedures are provided in the instruction manual. The wheat elongation factor TaEF-1α gene (GenBank accession number: M90077.2) was used as an internal reference gene, and primers were designed based on the TabHLH168 gene sequence for qRT-PCR analysis.

[0035] The primers designed for qRT-PCR analysis are 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] Primers must be tested for specificity and amplification efficiency (≥90%) of their amplification products before use. Using TB... Premix Ex Taq TM Using a Takara (Beijing, China) and an ABI QuantStudio 6 Flex quantitative PCR instrument (ThermoFisher, America), qRT-PCR was performed with cDNA from each sampling site as a template, following the instruction manual. Each reaction was performed in triplicate. The average Ct value was used, and the experimental data were analyzed using the Delta-Ct method. The Ct value from the 0h sampling site was set as a control (value of 1) to determine the relative expression level of the TabHLH168 gene at each sampling site.

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

[0042] Example 3: Validation of resistance by overexpression of the transcription factor protein encoding gene TabHLH168

[0043] 1. Preparation of wheat transformant lines:

[0044] Based on the results of Example 1, the target gene was obtained. Full-length primers carrying the recombinant fragment were designed to amplify the full-length fragment of the TabHLH168 gene, which was then ligated into the pOE3 plant expression vector via 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] In the common spring wheat variety JW1, a stable overexpression transformant line was obtained using Agrobacterium-mediated transformation. This transformant line overexpressed the TabHLH168 gene. Genomic DNA was extracted from the T3 generation transgenic wheat and the control wheat (JW1) using the CTAB method. Insertion fragments were detected and sequenced using the pOE-F / R primers.

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

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

[0051] Two stably heritable transformant lines were obtained through experiments, designated H168-L3 and H168-L6, respectively. The relative expression levels of the TabHLH168 gene transcript within the transformant lines were detected according to the method described in Example 2. Figure 3 As shown, the TabHLH168 gene was significantly overexpressed in both the H168-L3 and H168-L6 lines compared to wild-type wheat (JW1).

[0052] 2. Disease resistance analysis of transgenic wheat:

[0053] The *Fusarium graminearum* spore suspension was prepared according to the method in Example 2. The two transgenic lines H168-L3 and H168-L6 and the wild-type JW1 variety were disinfected with 1% sodium hypochlorite for 5 min, rinsed three times with sterile water, and immersed in the prepared conidia suspension for 5 min. Ten seeds were placed in 9 cm diameter petri dishes lined with three layers of filter paper, and cultured in a constant temperature and light incubator at 22℃ for 4 days. The spore suspension was then inoculated at the base of wheat stems, 20 μL per plant, and cultured in a humidity incubator for 5 days before disease investigation.

[0054] Grading standards:

[0055] Grade 0: No browning symptoms;

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

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

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

[0059] Level 4: The length of browning 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 value at each level) / (total number of plants surveyed × highest representative value) × 100%.

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

[0062] Therefore, this invention utilizes the above-mentioned transcription factor TabHLH168 protein in wheat disease resistance, overexpressing the gene encoding TabHLH168 protein to obtain transgenic wheat with stable heritability and resistance to wheat stem base rot. This provides a new solution for carrying out wheat disease resistance breeding work 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 solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

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

2. Overexpression of the TabHLH168 protein (amino acid sequence shown in SEQ ID NO.1) in wheat plants enhances wheat's resistance to Fusarium pseudobulbarum. Fusarium pseudograminearum Resistance to wheat stem rot caused by the disease.

2. The application of a recombinant vector containing the gene sequence shown in SEQ ID NO.2 in the breeding of wheat resistant to stem base rot, characterized in that: The recombinant vector is a plant overexpression vector, and stem base rot is caused by Fusarium pseudobulb. Fusarium pseudograminearum cause.

3. A method for breeding wheat varieties resistant to stem base rot, characterized in that: The recombinant vector as described in claim 2 was constructed, and then introduced into wheat plants to obtain a new transgenic wheat variety; stem base rot is caused by Fusarium pseudobulb. Fusarium pseudograminearum cause.

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