Application of TaSYT5 gene or related biological materials in regulating plant resistance to stem rot
By inhibiting the expression of the TaSYT5 gene and silencing the TaSYT5 gene in plants using RNAi technology, the problem of unsatisfactory control of stem base rot was solved, and plant varieties with resistance to stem base rot were cultivated.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHENZHEN RESEARCH INSTITUTE OF NORTHWEST A & F UNIVERSITY
- Filing Date
- 2025-10-10
- Publication Date
- 2026-05-26
AI Technical Summary
Existing technologies are not effective in controlling stem base rot, and there is a lack of effective means of regulating susceptible genes, resulting in insufficient resistance of plants such as wheat to stem base rot.
By suppressing the expression of the TaSYT5 gene, the TaSYT5 gene was silenced in plants using RNAi-mediated gene editing technology, thereby improving the plant's resistance to stem rot. The RNAi silencing vector was introduced into the plant using Agrobacterium-mediated genetic transformation to reduce the expression and activity of the TaSYT5 protein.
It significantly enhanced the plant's resistance to stem base rot, provided new genetic resources and disease-resistant materials, and bred plant varieties resistant to stem base rot.
Smart Images
Figure CN120905252B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural biotechnology, specifically involving TaSYT5 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, which can provide new gene resources and disease-resistant materials for breeding stem rot resistant varieties. Summary of the Invention
[0005] Stem base rot is caused by fungi including Phytophthora, Pythium, and Rhizoctonia. Among these, Phytophthora and Pythium, along with other fungi, infect plants, severely impacting plant growth. Conventional control methods are not ideal. Susceptibility genes can regulate the disease course before, during, and after pathogen invasion. Screening and researching susceptibility genes is a key approach to improving plant resistance to stem base rot. This invention aims to provide a plant susceptibility-related gene and study its interaction with plant resistance to stem base rot, providing new gene resources and resistant materials for plant resistance to stem base rot and for breeding resistant varieties.
[0006] On the one hand, the present invention provides TaSYT5 The application of genes or related biological materials in regulating plant resistance to stem base rot, the aforementioned TaSYT5 The nucleotide sequence of the gene is shown in SEQ ID No:1;
[0007] The relevant biomaterial contains the above. TaSYT5 Expression cassettes of gene nucleic acid molecules, recombinant vectors, recombinant microorganisms or transgenic cell lines or TaSYT5 The TaSYT5 protein is encoded by a gene.
[0008] The above-mentioned encoding TaSYT5 Expression cassettes of gene nucleic acid molecules ( TaSYT5 Gene expression cassettes (GCs) are devices that can express gene information in host cells. TaSYT5 The DNA, which can include not only the starter TaSYT5 The promoter of transcription may also include a terminator. TaSYT5 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... TaSYT5Recombinant 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 region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation control signals and start codons are widely available and 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 vectors used can be processed. This can involve adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic marker genes (such as the nptII gene for resistance to kanamycin and related antibiotics, the bar gene for resistance to the herbicide phosphinic acid, the hph gene for resistance to the antibiotic hygromycin, the dhfr gene for resistance to methotrexate, and the EPSPS gene for resistance to glyphosate), or chemical reagent resistance marker genes (such as herbicide resistance genes), and mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. From a safety perspective, transgenic plants can be screened directly under stress 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 by: inhibiting the expression of the TaSYT5 gene in the plant, thereby increasing the plant's resistance to stem base rot.
[0014] Furthermore, the inhibition TaSYT5 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 823 to 1064 of SEQ ID No:1 from the 5' end.
[0016] Furthermore, TaSYT5 The protein encoded by the gene is TaSYT5 protein, and the amino acid sequence of TaSYT5 protein is shown in SEQ ID No:2.
[0017] To facilitate the purification of the TaSYT5 protein, tags 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 TaSYT5 protein can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically.
[0021] Furthermore, TaSYT5 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. TaSYT5 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. TaSYT5 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 the pWMB110-TaSYT5-RNAi silencing vector into recipient plants to inhibit... TaSYT5 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: by editing... TaSYT5Genes were used to reduce the expression level and / or activity of the TaSYT5 protein in plants, resulting in plant varieties 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. TaSYT5 Genes play a negative regulatory role in the defense response of plants against stem rot pathogens, specifically by suppressing susceptibility-related genes. TaSYT5 Gene expression and reduced TaSYT5 protein expression can improve the plant's resistance to stem rot. TaSYT5 Genes or their biological materials can be used to improve plant resistance to stem base rot.
[0028] (2) This invention provides a method for breeding plant varieties resistant to stem base rot. This method utilizes RNAi-mediated gene editing technology to silence the aforementioned susceptibility-related genes within the plant. TaSYT5 Genes that enhance plant resistance to stem base rot. It has been verified that the gene obtained using the method of this invention... TaSYT5 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 TaSYT5 A schematic diagram of gene sequences and RNAi-mediated stable silencing fragments, where the sequences corresponding to the red underlined parts are schematic diagrams of RNAi-mediated stable silencing fragments.
[0030] Figure 2 This is a schematic diagram of the nucleotide sequence of the RNAi hairpin structure.
[0031] Figure 3 for TaSYT5 A schematic diagram illustrating the molecular identification of gene-silenced plants. A represents the T0 generation. TaSYT5 Positive identification results of gene-silenced plants; B is a positive T0 generation. TaSYT5 In gene-silenced plants TaSYT5 Relative gene expression levels; C represents T2 generation. TaSYT5 Positive identification results of gene-silenced plants; D indicates positive T2 generation. TaSYT5 In gene-silenced plants TaSYT5 Relative gene expression levels; E indicates T2 generation positivity. TaSYT5 Agronomic traits of gene-silenced plants before inoculation.
[0032] Figure 4 for TaSYT5Schematic diagram of phenotypic identification of resistance to wheat stem rot in gene-silenced plants. 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 TaSYT5 protein and its encoding gene. TaSYT5 Acquisition of genes.
[0038] Take normally grown 7-day-old wheat Fieler seedlings, quick-freeze them with liquid nitrogen, and store them at -80℃ for later use.
[0039] Total RNA was extracted from wheat Fieler 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, PCR amplification was performed using primer pairs TaSYT5-F and TaSYT5-R to obtain... TaSYT5 The specific sequences of the gene and primer pair are as follows:
[0040] TaSYT5-F (SEQ ID No:3):5'-ATGGCGTCTCTCTCGGCGCCCT-3';
[0041] TaSYT5-R (SEQ ID No: 4):5'-TCACTGGTCCCTGTCACGATCAC-3'.
[0042] TaSYT5 The PCR products of the gene were detected by 1.0% agarose gel electrophoresis. Those that passed the test... TaSYT5 The PCR product of the gene was sent to a biotechnology company for sequencing. The sequencing results showed that... TaSYT5 The gene is 1689 bp in size, and its nucleotide sequence is shown in SEQ ID No:1. It is named... TaSYT5The gene encodes a protein whose amino acid sequence is shown in SEQ ID No:2, and the protein is named TaSYT5 protein.
[0043] Example 2
[0044] This embodiment is for suppression TaSYT5 Application of gene expression in regulating plant resistance to stem rot.
[0045] (1) TaSYT5 Obtaining gene-silenced plants
[0046] The composition of the hairpin structure:
[0047] in accordance with TaSYT5 The nucleotide sequence of a hairpin structure was designed from gene sequences for RNAi-mediated RNA injection. TaSYT5 Gene silencing, TaSYT5 A schematic diagram of gene sequences and RNAi-mediated stable silencing fragments is shown below. Figure 1 As shown. The hairpin structure designed above was synthesized. This hairpin structure consists of a 242bp silencing fragment, a 146bp intron fragment, and a 242bp inverse complementary silencing fragment. The nucleotide sequence of the hairpin structure is shown below. Figure 2 As shown, the hairpin structure was synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd.
[0048] Construction of the pWMB110-TaSYT5-RNAi silencing vector:
[0049] The hairpin structure was introduced into the pWMB110 vector using homologous recombination technology to form the pWMB110-TaSYT5-RNAi silencing vector.
[0050] Agrobacterium-mediated genetic transformation of wheat:
[0051] The constructed gene-silencing recombinant vector pWMB110-TaSYT5-RNAi was transformed into immature embryos of the wheat variety Fielder via Agrobacterium-mediated genetic transformation. Following differentiation, selection, regeneration, and rooting, regenerated plants were obtained. The wheat genetic transformation was completed 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] TaSYT5 Molecular identification of gene-silenced plants:
[0053] Test plants: 9 T0 generation regenerated plants obtained in the previous step, and Fielder (as a reference plant for the regenerated plants).
[0054] Genomic DNA was extracted from the leaves of the tested plants. PCR was used to detect the selection gene bar and identify positive T0 generation wheat plants that had TaSYT5 gene silencing. The positive T0 generation plants Ri1, Ri7, and Ri8 were retained, and T2 generation TaSYT5 gene-silencing plants were obtained through self-pollination. DNA was also extracted from the T2 generation TaSYT5 gene-silencing plants, and PCR was used to detect the selection gene bar to identify positive TaSYT5 gene-silencing plants. The silencing level of TaSYT5 in the positive TaSYT5 gene-silencing plants was detected by qRT-PCR using the TaSYT5-F and TaSYT5-R primer pairs. The identification results are as follows: Figure 3 As shown, all 10 TaSYT5-Ri1-T2, 10 TaSYT5-Ri7-T2, and 10 TaSYT5-Ri8-T2 strains tested positive. TaSYT5 Gene-silenced plants.
[0055] (2) TaSYT5 Analysis of stem base rot resistance in gene-silenced plants
[0056] Test plants: Fielder plants, TaSYT5-Ri1-T2 plants, TaSYT5-Ri7-T2 plants, and TaSYT5-Ri8-T2 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 every day) 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 grain: soil substrate: water = 1:10:1 (volume ratio) and put it 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 disease resistance index of the tested plants to stem base rot fungus. The results show... TaSYT5Gene-silenced plants TaSYT5-Ri1-T2, TaSYT5-Ri7-T2, and TaSYT5-Ri8-T2 showed significantly lower disease indices compared to the wild-type Fielder material. This indicates... TaSYT5 Gene-silenced plants exhibited strong resistance to stem rot pathogens. This indicates... TaSYT5 It is an important gene associated with susceptibility to stem base rot. TaSYT5 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. TaSYT5 The application of genes in improving wheat resistance to stem base rot is characterized by, The pathogen causing the stem base rot is *Fusarium graminearum* (Pseudomonas graminearum). Fusarium pseudograminearum ), the TaSYT5 The nucleotide sequence of the gene is shown in SEQ ID No:1; in the wheat, the silenced gene... TaSYT5 Gene expression enhances the wheat's resistance to stem rot.
2. The application according to claim 1, characterized in that, The silence TaSYT5 Gene expression is achieved by introducing RNA interference fragments and RNA interference vectors into the wheat.
3. The application according to claim 2, characterized in that, The RNA interference vector contains nucleotides from position 823 to 1064 of SEQ ID No:1 from the 5' end.
4. The application according to any one of claims 1-2, characterized in that, TaSYT5 The protein encoded by the gene is TaSYT5 protein, and the amino acid sequence of TaSYT5 protein is shown in SEQ ID No:
2.
5. A method for improving wheat resistance to stem rot, comprising silencing receptor wheat TaSYT5 Gene expression, the TaSYT5 The nucleotide sequence of the gene is shown in SEQ ID No:1, and the pathogen of the stem base rot is *Fusarium graminearum*. Fusarium pseudograminearum ).
6. A method for breeding wheat varieties resistant to stem base rot, comprising silencing recipient wheat... TaSYT5 Gene expression was used to obtain wheat with gene-silenced resistance to stem base rot, the pathogen of which is *Fusarium graminearum* (Pseudomonas graminearum). Fusarium pseudograminearum ), the TaSYT5 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: silencing... TaSYT5 Genes were used to reduce the expression level and / or activity of the TaSYT5 protein in wheat to obtain wheat varieties resistant to stem base rot, wherein the pathogen of stem base rot is Fusarium pseudobulb (…). Fusarium pseudograminearum ), the TaSYT5 The nucleotide sequence of the gene is shown in SEQ ID No:1.