Application of tabzip69 gene or related biological material in regulating resistance of plants to stem base rot

Abstract

The application belongs to the field of agricultural biotechnology, and particularly relates to a plant disease-related gene TabZIP69 or application of related biological materials thereof in regulating resistance of a plant to stem base rot. The application provides a plant disease-related gene TabZIP69 , an amino acid sequence of a protein encoded by the gene is shown as SEQ ID No: 2, and a nucleotide sequence of the plant disease-related gene TabZIP69 is shown as SEQ ID No: 1. The application performs gene stable silencing in Fielder wild type material to obtain a gene silencing plant showing resistance to plant stem base rot. TabZIP69 The application determines that TabZIP69 the gene plays a negative regulation role in plant resistance to stem base rot, and uses the negative regulation factor to create a plant material resistant to stem base rot, thereby providing a new gene and germplasm resource for plant breeding for resistance to stem base rot.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically involving TabZIP69 Application of genes or related biological materials in regulating plant resistance to stem base rot. Background Technology

[0002] Stem base rot is one of the major diseases affecting crop production. It is primarily caused by various pathogens, including *Fusarium graminearum* (…). Fusarium pseudograminearum Fusarium graminearum ( ), Fusarium graminearum F. graminearum ), Fusarium oxysporum ( F. culmorum ), Fusarium tumefaciens ( F. asiaticum Fusarium solani ( ), Fusarium solani F. avenaceum Fusarium oxysporum ( F. oxysporum ) and Fusarium equisetifolium ( F. equiseti (e.g., wheat). Taking wheat as an example, the pathogen mainly infects the base of the wheat stem. The pathogen germinates as hyphae or spores on the soil surface, invades the wheat through the roots and coleoptile, further enters the internodes under the crown and leaf sheaths, and then penetrates the stem epidermal tissue through stomata. As the wheat grows and develops, after the pathogen successfully colonizes the base of the wheat stem, it blocks the vascular tissue, thereby blocking the transport of water and nutrients inside the wheat plant, ultimately leading to lodging, small ears and shriveled grains, seriously affecting the yield and quality of wheat.

[0003] Pathogen infection in plants triggers a defensive response. Simultaneously, pathogens utilize host components to regulate their own growth, infection structure differentiation, negatively regulate the plant's immune response, and absorb nutrients from the host, thus causing host susceptibility. All host genes that promote pathogen infection and support affinity interactions are considered susceptibility genes. In the early stages of pathogen infection, susceptibility genes function by promoting host recognition of pathogen invasion; during the invasion phase, they increase susceptibility by negatively regulating the host's immune response; once successful invasion and the establishment of a parasitic relationship, the pathogen utilizes host nutrients to meet its own metabolic and growth needs, promoting susceptibility. For example, sugar transporters (OsSWEET11 and OsSWEET13), aspartate metabolism pathway homoserine kinase genes (HSK), and aspartate kinase (AK2) mediate host susceptibility to pathogens. Silencing susceptibility genes can limit the pathogen's ability to infect and cause disease, thereby enhancing the host's resistance.

[0004] Therefore, it is of great significance to find susceptibility genes, regulate these susceptibility genes, and explore the function of these susceptibility genes in the interaction process with stem rot fungus. This can provide new gene resources and disease-resistant materials for breeding stem rot resistant varieties. Summary of the Invention

[0005] The present invention aims to provide a plant susceptibility-related gene, study its interaction with plant resistance to stem rot, and provide new gene resources and disease-resistant materials for plant resistance to stem rot and the breeding of disease-resistant varieties.

[0006] On the one hand, the present invention provides TabZIP69 The application of genes or related biological materials in regulating plant resistance to stem base rot, the aforementioned TabZIP69 The nucleotide sequence of the gene is shown in SEQ ID No:1;

[0007] The relevant biomaterial contains the above. TabZIP69 Expression cassettes of gene nucleic acid molecules, recombinant vectors, recombinant microorganisms or transgenic cell lines or TabZIP69 The TabZIP69 protein encoded by the gene.

[0008] The above-mentioned encoding TabZIP69 Expression cassettes of gene nucleic acid molecules ( TabZIP69 Gene expression cassettes (GCs) are devices that can express gene information in host cells. TabZIP69 The DNA, which can include not only the starter TabZIP69 The promoter of transcription may also include a terminator. TabZIP69 Transcription terminators. Further, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters; tissue-, organ-, and development-specific promoters; and inducible promoters. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminator.

[0009] Existing expression vectors can be used to construct structures containing the aforementioned... TabZIP69Recombinant vectors for gene expression cassettes. These plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microbombardment. Examples include pWMB110, pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The plant expression vectors may also contain the 3' untranslated region of a foreign gene, i.e., a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal guides the addition of polyadenylated acid to the 3' end of the mRNA precursor; similar functions exist in the 3' untranslated regions of Agrobacterium crown gall-inducing (Ti) plasmid genes (such as the nosine synthase gene) and plant genes (such as the soybean storage protein gene). When constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as by adding genes encoding enzymes or luminescent compounds that can be expressed in plants (e.g., genes encoding enzymes that produce color changes). GUS Genes, luciferase genes, etc.), and antibiotic marker genes (such as those conferring resistance to kanamycin and related antibiotics). nptII Genes, including the bar gene which confers resistance to the herbicide phosphinic acid, and the bar gene which confers resistance to the antibiotic hygromycin. hph Genes, and the genes that confer resistance to methotrexate dhfr Genes that confer resistance to glyphosate EPSPS Genes such as herbicide-resistant marker genes or mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose can be used. From a safety perspective, transgenic plants can be directly selected by stress screening without adding any selective marker genes.

[0010] The aforementioned vectors can be plasmids, granules, bacteriophages, or viral vectors.

[0011] The aforementioned microorganisms can be yeast, bacteria, algae, or fungi, such as Agrobacterium.

[0012] The aforementioned transgenic plant cell lines do not include propagation material.

[0013] Furthermore, the regulation of plant resistance to stem base rot is manifested in the following way: in the plant, inhibition TabZIP69 Gene expression enhances the plant's resistance to stem base rot.

[0014] Furthermore, the inhibition TabZIP69 Gene expression is achieved by introducing RNA interference fragments, RNA interference vectors, homologous recombination fragments, homologous recombination vectors, or gene editing tools into the plant.

[0015] Furthermore, the RNA interference vector contains nucleotides from position 322 to 487 of SEQ ID No:1 from the 5' end.

[0016] Furthermore, TabZIP69 The protein encoded by the gene is TabZIP69 protein, and the amino acid sequence of TabZIP69 protein is shown in SEQ ID No:2.

[0017] To facilitate the purification of the TabZIP69 protein, a tag as shown in Table 1 can be attached to the amino or carboxyl terminus of the protein composed of the amino acid sequence shown in SEQ ID No:2.

[0018] Table 1 shows the sequence of labels.

[0019]

[0020] The TabZIP69 protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.

[0021] Furthermore, TabZIP69 The application of genes or related biological materials in regulating plant resistance to stem base rot, wherein the plant is a monocotyledonous or dicotyledonous plant, the monocotyledonous plant is a grass, the grass is a member of the Poaceae family, and the member of the Poaceae family is wheat.

[0022] On the other hand, the present invention provides a method for improving plant resistance to stem base rot, including inhibiting the activity of the receptor plant in the stem base rot. TabZIP69 Gene expression.

[0023] Furthermore, the present invention also provides a method for cultivating plant varieties resistant to stem base rot, comprising inhibiting the growth of stem base rot in recipient plants. TabZIP69 Gene expression was used to obtain gene-silenced plants resistant to stem base rot.

[0024] Furthermore, the method for cultivating plant varieties resistant to stem base rot includes using Agrobacterium-mediated transformation of pWMB110- TabZIP69 -RNAi silencing vectors, when transformed into recipient plants, inhibit... TabZIP69 Gene expression was used to obtain gene-silenced plants resistant to stem base rot.

[0025] Furthermore, the method for cultivating plant varieties resistant to stem base rot includes the following steps: through stable silencing TabZIP69 Genes were used to reduce the expression level and / or activity of the TabZIP69 protein in plants, resulting in plant strains resistant to stem rot.

[0026] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0027] (1) This invention discovers disease-related genes through gene function research. TabZIP69 Genes play a negative regulatory role in the defense response of plants against stem rot pathogens, specifically by suppressing susceptibility-related genes. TabZIP69 Increasing gene expression and reducing the expression of TabZIP69 protein can improve the plant's resistance to stem rot. TabZIP69 Genes or their biological materials can be used to improve plant resistance to stem rot.

[0028] (2) This invention provides a method for breeding plant varieties resistant to stem base rot. This method utilizes RNAi-mediated gene silencing technology to silence the aforementioned susceptibility-related genes within the plant. TabZIP69 Genes that enhance plant resistance to stem base rot. Verification has shown that the method obtained using this invention... TabZIP69 Gene-silenced plants exhibit resistance to stem base rot. This invention provides a new technical approach for breeding plant varieties resistant to stem base rot from a molecular biology perspective, effectively solving the technical problems of this invention. Attached Figure Description

[0029] Figure 1 Provided for embodiments of the present invention TabZIP69 A schematic diagram of the gene sequence and the stable silencing fragment mediated by RNAi.

[0030] Figure 2 This is a schematic diagram of the nucleotide sequence of the RNAi hairpin structure.

[0031] Figure 3 for TabZIP69 Image showing the identification results of gene-silenced plants; Figure 3 A in the middle is T0 generation TabZIP69 Image showing PCR identification results of gene-silenced plants; Figure 3 B is the T0 generation TabZIP69 In gene-silenced plants and positive plants TabZIP69 The relative expression level of genes; Figure 3 The middle C is the T3 generation. TabZIP69 Gene-silenced plants TabZIP69 -Ri1-T3、 TabZIP69 -Ri3-T3 and TabZIP69-PCR identification results of Ri4-T3; Figure 3 D is the T3 generation TabZIP69 Gene-silenced plants and positive plants TabZIP69 -Ri1-T3、 ​ -Ri3-T3 and ​ -Ri4-T3 ​ The relative expression level of genes; ​ E in the middle is a T3 generation positive. ​ Agronomic traits of gene-silenced plants before inoculation.

[0032] ​ for ​ Schematic diagram of phenotypic identification of resistance to wheat stem rot in gene-silenced plants; ​ Figure A shows a schematic diagram of the resistance phenotype of the tested plants to stem rot fungus. ​ Figure B shows the statistical results of the biomass of the tested plants against the resistant fungi of stem rot. Detailed Implementation

[0033] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0034] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0035] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available; and unless otherwise specified, the percentages in the following embodiments are all mass percentages.

[0036] Example 1

[0037] This example describes the TabZIP69 protein and its encoding gene. ​ Acquisition of genes.

[0038] Take normally grown 7-day-old wheat Fielder seedlings, quick-freeze them with liquid nitrogen, and store them at -80℃ for later use.

[0039] Total RNA was extracted from wheat Fielder seedlings using the Trizol method (Tiangen Biotech (Beijing) Co., Ltd.). First-strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was synthesized using the SMART method. Using the cDNA as a template, primer pairs were used... ​ -F and ​ -R, obtained by PCR amplification ​ The specific sequences of the gene and primer pair are as follows:

[0040] ​ -F (SEQ ID No:3):5'-ATGGCAGATGCTAGCTCAAGGACTG-3';

[0041] ​ -R (SEQ ID No: 4):5'-TTATTCCCTGGGCCTAGCAAGCCAC-3'.

[0042] ​ The PCR products of the gene were detected by 1.0% agarose gel electrophoresis. Those that passed the test... ​ The PCR product of the gene was sent to a biotechnology company for sequencing. The sequencing results showed that... ​ The gene is 1001 bp in size, and its nucleotide sequence is shown in SEQ ID No:1. It is named... ​ The gene encodes a protein whose amino acid sequence is shown in SEQ ID No:2, and the protein is named TabZIP69 protein.

[0043] Example 2

[0044] This embodiment is for suppression ​ Application of gene expression in regulating plant resistance to stem rot.

[0045] (1) ​ Obtaining gene-silenced plants

[0046] Composition of the hairpin structure:

[0047] in accordance with ​ The nucleotide sequence of a hairpin structure was designed from gene sequences for RNAi-mediated RNA injection. ​ Gene silencing, ​ A schematic diagram of gene sequences and RNAi-mediated stable silencing fragments is shown below. ​ As shown. The hairpin structure designed above was synthesized. This hairpin structure consists of a 166bp silencing fragment, a 146bp intron fragment, and a 166bp inverse complementary silencing fragment. The nucleotide sequence of the hairpin structure is shown below. ​ As shown, the hairpin structure was synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd.

[0048] pWMB110- ​ - Construction of RNAi silencing vector:

[0049] The synthesized hairpin structure sequence was ligated into the pWMB110 vector using homologous recombination technology (one-step rapid cloning technology) to form pWMB110- ​ -RNAi silencing vector.

[0050] Agrobacterium-mediated genetic transformation of wheat:

[0051] The constructed gene silencing recombinant vector pWMB110- ​ RNAi was transformed into immature embryos of the wheat variety Fielder via Agrobacterium-mediated genetic transformation. The embryos then underwent differentiation, selection, regeneration, and rooting to ultimately obtain regenerated plants. The wheat genetic transformation was performed by the Genetic Transformation Platform of the National Key Laboratory of Crop Stress Resistance and High-Efficiency Production at Northwest A&F University. The public can commercially contact this platform to complete the transformation process.

[0052] ​ Molecular identification of gene-silenced plants:

[0053] Test plants: ​ Gene-silenced plants and Fielder plants (control).

[0054] Genomic DNA was extracted from the leaves of the tested plants. Seven silent DNA lines were identified using PCR technology. ​ T0 generation wheat plants were selected as positive for the gene. The T0 generation plants Ri1, Ri3, and Ri4 were retained, and T2 and T3 generations were obtained through self-pollination. ​ Gene-silenced plants, for the T3 generation ​ DNA was also extracted from gene-silenced plants, and positive results were identified using PCR technology. ​ Gene-silenced plants, positive ​ Primers for identifying gene-silenced plants were all used. ​ -F and ​ -R primer pair. Identification results are as follows: ​ As shown, ​ A in the middle is T0 generation ​ Image showing PCR identification results of gene-silenced plants; Fielder lanes represent wild-type wheat, H2O lanes represent negative control water, Marker lanes represent molecular weight, and Ri1-Ri9 lanes represent generation T0. ​ Nine gene-silenced plants were analyzed, and the results showed that, except for Ri2 and Ri5, the remaining seven T0 generations... ​ The selection gene was detected in all gene-silenced plants. ​ The gene fragment (435bp) indicates that 7 T0 generation strains ​ Gene-silenced plants and positive plants. These are respectively... ​ -Ri1-T0 (Ri1) ​ -Ri3-T0 (Ri3) ​ -Ri4-T0 (Ri4) ​ -Ri4-T0 (Ri6) ​ -Ri7-T0 (Ri7) ​ -Ri8-T0 (Ri8) and ​ -Ri9-T0 (Ri9). ​B is the T0 generation ​ The relative expression level of the TabZIP69 gene in gene-silenced positive plants; the results showed that in the T0 generation... ​ Comparison of gene-silenced positive plants Ri1, Ri3, Ri4, Ri6, Ri7, Ri8, and Ri9 with wild-type wheat Fielder ​ Gene expression levels decreased significantly. ​ The middle C is the T3 generation. ​ Gene-silenced plants ​ -Ri1-T3、 ​ -Ri3-T3 and ​ PCR identification results of Ri4-T3 showed that 10 strains ​ -Ri1-T3, 10 strains ​ -Ri3-T3 and 10 plants ​ -Ri4-T3 both express the selection gene ​ All of them ​ Gene-silenced plants are positive plants. ​ D is the T3 generation ​ Gene-silenced plants and positive plants ​ -Ri1-T3、 ​ -Ri3-T3 and ​ -Ri4-T3 ​ The relative expression levels of genes indicate that the T3 generation... ​ Gene-silenced plants and positive plants ​ -Ri1-T3、 ​ -Ri3-T3 and ​ -Ri4-T3 vs. Fielder ​ The relative expression level of the gene decreased significantly. ​ E in the middle is a T3 generation positive. ​ Agronomic trait diagram of gene-silenced plants before inoculation, T3 generation positive. ​ Gene-silenced plants and Fielder plants grew well.

[0055] (2) ​ Analysis of stem base rot resistance in gene-silenced plants

[0056] Test plants: Fielder plants, ​ -Ri1-T3 plants, TabZIP69 -Ri3-T3 plants, TabZIP69 -Ri4-T3 plants.

[0057] The steps for identifying stem base rot resistance in the above-mentioned tested plants are as follows:

[0058] Conidium culture: Wild-type strain of *Fusarium graminearum*, one of the pathogens of wheat stem rot, was selected as the strain. The strain was activated by culturing on PDA solid medium. A piece of solid containing the stem rot strain was taken from the above medium using a disc sampler and placed in 250g of millet solid medium that had been washed, boiled, dried and sterilized. The medium was cultured at 25℃ for 6-7 days (the medium was shaken daily) to obtain diseased grains containing conidia of the wheat stem rot pathogen, which were then used for later use.

[0059] Diseased grain mixed with soil inoculation method: Before inoculation, press the soil substrate of the test plant down to 1cm deep from the edge, then mix the diseased grains, soil substrate and water in a ratio of 1:10:1 (by volume) and put them into the flower pot to complete the inoculation.

[0060] Twenty-one days after inoculation, the disease incidence at the base of wheat stems was investigated and statistically analyzed. The disease index was used to evaluate the stem base rot resistance of the tested plants.

[0061] Test results are as follows Figure 4 As shown, Figure 4 Figure A shows a schematic diagram of the resistance phenotype of the tested plants to stem rot fungus. Figure 4 Figure B shows the statistical results of the biomass of the tested plants against the resistant fungi of stem rot pathogen. The results show... TabZIP69 Gene-silenced plants TabZIP69 -Ri1-T3、 TabZIP69 -Ri3-T3 and TabZIP69 -Ri4-T3 showed a significant decrease in fungal biomass compared to the wild-type material Fielder. This indicates... TabZIP69 Gene-silenced plants exhibited strong resistance to stem rot pathogens. This indicates... TabZIP69 It is an important gene associated with susceptibility to stem base rot. TabZIP69 The gene plays a negative regulatory role in the interaction between plants and stem base rot. Inhibiting the expression of this gene can improve the plant's resistance to stem base rot, and inhibiting the expression of this gene can cultivate plant varieties resistant to stem base rot.

Claims

1. Inhibition TabZIP69 Use of a biological material related to the inhibition of gene expression in increasing the resistance of wheat to stem base rot, characterized in that, The TabZIP69 The nucleotide sequence of the gene is shown as SEQ ID No: 1, and the pathogen of the stem base rot is Pseudocercosporella herpotrichoides (DID Fusarium pseudograminearum ).

2. Use according to claim 1, characterized in that, The inhibition TabZIP69 Expression of the gene is achieved by introducing into the wheat an RNA interference fragment, an RNA interference vector, a homologous recombination fragment, a homologous recombination vector, or a gene editing tool.

3. Use according to claim 2, characterized in that, The RNA interference vector contains nucleotides 322-487 from the 5' end of SEQ ID No:

1.

4. The use according to any one of claims 1 to 2, characterized in that, TabZIP69 The protein encoded by the gene is a TabZIP69 protein, and the amino acid sequence of the TabZIP69 protein is shown as SEQ ID No:

2.

5. A method for improving wheat resistance to stem rot, comprising inhibiting receptor wheat... TabZIP69 Gene expression, the pathogen of the stem base rot is Fusarium pseudobulb ( Fusarium pseudograminearum ), the TabZIP69 The nucleotide sequence of the gene is shown in SEQ ID No:

1.

6. A method of breeding a wheat variety resistant to stem base rot, comprising suppressing the expression of a gene in a recipient wheat, to obtain a gene-silenced wheat resistant to stem base rot, the pathogen of which is Pseudocercosporella herpotrichoides (Ditmar) Roberge ex Desm. TabZIP69 Fusarium pseudograminearum TabZIP69 The nucleotide sequence of the gene is shown in SEQ ID No: 1.​​ 7. A method for breeding wheat varieties resistant to stem base rot, comprising the following steps: through stable silencing... TabZIP69 Genes were used to reduce the expression level and / or activity of the TabZIP69 protein in wheat, resulting in a wheat line resistant to stem base rot, wherein the pathogen of stem base rot is *Fusarium graminearum*. Fusarium pseudograminearum ), the TabZIP69 The nucleotide sequence of the gene is shown in SEQ ID No:1.

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