Application of TaSYT5 gene or related biological materials thereof in regulating and controlling stem rot resistance of plants

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to application of a plant susceptibility related gene TaSYT5 or a related biological material thereof to regulation and control of plant basal stem rot resistance. The amino acid sequence of the encoding protein of the plant disease related gene TaSYT5 provided by the invention is as shown in SEQ ID No: 2, and the nucleotide sequence of the plant disease related gene TaSYT5 is as shown in SEQ ID No: 1. According to the invention, gene editing is carried out in a Fielder wild type material, so that a TTaSYT5 gene silencing plant with resistance to plant basal stem rot is obtained. It is determined that the TaSYT5 gene plays a negative regulation role in plant stem rot resistance, a stem rot resistant plant material is created by using the negative regulation factor, and a new gene and germplasm resource are provided for plant stem rot resistant breeding.

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; 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.

[0007] 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.

[0008] Existing expression vectors can be used to construct structures containing the aforementioned... TaSYT5Recombinant vectors for gene expression cassettes. The plant expression vectors include binary Agrobacterium vectors and vectors useful for plant microprojectile bombardment, etc. Such as pWMB110, pAHC25, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1305, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA), etc. The plant expression vectors can also contain a 3' untranslated region of a foreign gene, i.e. containing a polyadenylation signal and any other DNA segment involved in mRNA processing or gene expression. The polyadenylation signal can direct polyadenylation to the 3' end of the mRNA precursor, such as the untranslated region of the 3' end of the transcription of the Agrobacterium Ti plasmid genes (such as the nopaline synthase gene Nos), plant genes (such as the soybean storage protein gene), etc. When using the gene construction plant expression vectors of the present application, enhancers can also be used, including translation enhancers or transcription enhancers, which can be ATG start codon or adjacent regions start codon, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is wide, which can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that can be expressed in plants to produce color changes or luminescent compounds (GUS gene, luciferase gene, etc.), marker genes of antibiotics (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to herbicide phosphine, hph gene conferring resistance to antibiotic hygromycin, and dhfr gene conferring resistance to aminomethopterin, EPSPS gene conferring resistance to glyphosate), or chemical reagent resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase genes that provide the ability to metabolize mannose. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0009] The above-mentioned vectors can be plasmids, cosmids, bacteriophages or viral vectors.

[0010] The above-mentioned microorganisms can be yeasts, bacteria, algae or fungi, such as Agrobacterium.

[0011] The above-mentioned transgenic plant cell lines do not include propagation materials.

[0012] Further, the plant resistance to stem base rot is manifested as: in the plant, the expression of the TaSYT5 gene is inhibited, and the resistance of the plant to stem base rot is improved.

[0013] Further, the expression of the gene is inhibited by introducing an RNA interference fragment, an RNA interference vector, a homologous recombination fragment, a homologous recombination vector or a gene editing tool into the plant. TaSYT5

[0014] Further, the RNA interference vector contains nucleotides 823-1064 from the 5' end of SEQ ID No: 1.

[0015] Further, the gene encodes a protein of TaSYT5, and the amino acid sequence of the TaSYT5 protein is shown as SEQ ID No: 2. TaSYT5

[0016] In order to facilitate the purification of the TaSYT5 protein, a tag shown in Table 1 can be connected to the amino terminal or carboxyl terminal of the protein composed of the amino acid sequence shown in SEQ ID No: 2.

[0017] Table 1 is the sequence of the tag

[0018] The above-mentioned TaSYT5 protein can be artificially synthesized, or the encoding gene thereof can be synthesized first and then expressed biologically.

[0019] Further, the gene or the related biological material thereof is used for regulating the resistance of a plant to stem base rot, and the plant is a monocotyledon or a dicotyledon, the monocotyledon is a gramineous plant, the gramineous plant is a plant in the family Poaceae, and the plant in the family Poaceae is wheat. TaSYT5 In another aspect, the present application provides a method for improving the resistance of a plant to stem base rot, comprising inhibiting the expression of the gene in a receptor plant.

[0020] TaSYT5 In addition, the present application also provides a method for breeding a plant variety resistant to stem base rot, comprising inhibiting the expression of the gene in a receptor plant to obtain a gene-silenced plant resistant to stem base rot.

[0021] In addition, the present application also provides a method for breeding a plant variety resistant to stem base rot, comprising inhibiting the expression of the gene in a receptor plant to obtain a gene-silenced plant resistant to stem base rot. TaSYT5

[0022] Further, the method for breeding a plant variety resistant to stem base rot comprises transforming the pWMB110-TaSYT5-RNAi silencing vector into a receptor plant by Agrobacterium mediation to inhibit the expression of the gene and obtain a gene-silenced plant resistant to stem base rot. TaSYT5

[0023] Further, the method for breeding a plant variety resistant to stem base rot comprises the following steps: editing the gene in a receptor plant to obtain a gene-silenced plant resistant to stem base rot. TaSYT5 ​​​​​Genes were used to reduce the expression level and / or activity of the TaSYT5 protein in plants, resulting in plant varieties resistant to stem rot.

[0024] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: (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 rot.

[0025] (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. Verification has shown that the method obtained using 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

[0026] 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.

[0027] Figure 2 This is a schematic diagram of the nucleotide sequence of the RNAi hairpin structure.

[0028] 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.

[0029] Figure 4 for TaSYT5Schematic diagram of phenotype identification of gene-silenced plants against wheat sharp eyespot. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described below in conjunction with examples, but the present application is not limited to the following examples.

[0031] In order to enable those skilled in the art to better understand the technical solutions of the present application and to implement them, the present application will be further described below in conjunction with specific examples and drawings, but the examples are not limiting to the present application.

[0032] The experimental methods and detection methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified; and the % described in the following examples is all mass percentage unless otherwise specified.

[0033] Example 1 This example is TaSYT5 protein and its encoding gene TaSYT5 Obtaining of the gene.

[0034] Take normal growth 7-day-old wheat Fieler seedlings, freeze them with liquid nitrogen, and store them at -80℃ for standby.

[0035] Trizol method (Tiangen Biotech (Beijing) Co., Ltd.) is used to extract total RNA of wheat Fieler seedlings, and reverse transcriptase XL (AMV) is used for first-strand cDNA synthesis. cDNA is synthesized by SMART method, and primer pair TaSYT5-F and TaSYT5-R is used for PCR amplification to obtain the gene TaSYT5 The specific sequences of the primer pairs are as follows: TaSYT5-F (SEQ ID No: 3): 5'-ATGGCGTTCCTCTTCGGCGCCCT-3'; TaSYT5-R (SEQ ID No: 4): 5'-TCACTGGTCCCTGTCACGATCAC-3'.

[0036] TaSYT5 The PCR product of the gene is detected by 1.0% agarose gel electrophoresis, and the qualified TaSYT5 The PCR product of the gene is sent to a biological company for sequencing, and the sequencing result shows that TaSYT5 The gene is 1689 bp in size, and its nucleotide sequence is shown as SEQ ID No: 1, which is named as TaSYT5 The gene, and the amino acid sequence of the protein encoded by the gene is shown as SEQ ID No: 2, and the protein is named as TaSYT5 protein.

[0037] Example 2 This example is to inhibit TaSYT5 gene expression in regulating plant resistance to stem base rot.

[0038] (1) TaSYT5 Obtaining of gene silencing plants Synthesis of hairpin structure: According to TaSYT5 gene sequence design of hairpin structure of nucleotide sequence, for RNAi mediated TaSYT5 gene silencing, TaSYT5 sequence of the gene and RNAi mediated stable silencing fragment diagram as Figure 1 shown. Synthesis of the above designed hairpin structure. The hairpin structure contains 242 bp silencing fragment, 146 bp intron fragment and 242 bp reverse complementary silencing fragment. The nucleotide sequence of the hairpin structure is shown in Figure 2 , and the hairpin structure is synthesized by Beijing Aokedaisheng Biotechnology Co., Ltd.

[0039] Construction of pWMB110-TaSYT5-RNAi silencing vector: The above hairpin structure is introduced into pWMB110 vector by homologous recombination technology, forming pWMB110-TaSYT5-RNAi silencing vector.

[0040] Agrobacterium-mediated genetic transformation of wheat: The constructed gene silencing recombinant vector pWMB110-TaSYT5-RNAi is transformed into immature embryo of wheat variety Fielder by Agrobacterium-mediated genetic transformation, and then through differentiation, screening, regeneration and rooting, finally the regenerated plants are obtained. The genetic transformation of wheat is completed by the genetic transformation platform of the State Key Laboratory of Crop Stress Resistance and High Efficiency Production of Northwest A&F University, and the public can contact the platform for commercial transformation operation.

[0041] TaSYT5 Molecular identification of gene silencing plants: Test plants: 9 T0 generation regenerated plants obtained in the above step, Fielder (as a reference plant for regenerated plants).

[0042] Genomic DNA of leaves of the test plants was extracted. The gene Bar was detected by PCR technique to identify positive T0 generation wheat plants with silenced TaSYT5 gene, and T0 generation positive plants Ri1, Ri7 and Ri8 were reserved. T2 generation TaSYT5 gene silenced plants were obtained by self-crossing, and DNA of the T2 generation TaSYT5 gene silenced plants was also extracted. The gene Bar was detected by PCR technique to identify positive TaSYT5 gene silenced plants. The positive TaSYT5 gene silenced plants were detected by qRT-PCR to determine the silencing level of TaSYT5, and the qRT-PCR primers were TaSYT5-F and TaSYT5-R primer pairs. The identification results are shown in Table 1. Figure 3 As shown in Table 1, 10 TaSYT5-Ri1-T2 plants, 10 TaSYT5-Ri7-T2 plants and 10 TaSYT5-Ri8-T2 plants were all positive TaSYT5 gene silenced plants.

[0043] (2) TaSYT5 Analysis of stem base rot resistance of the gene silenced plants Test plants: Fielder plants, TaSYT5-Ri1-T2 plants, TaSYT5-Ri7-T2 plants and TaSYT5-Ri8-T2 plants.

[0044] The test plants were subjected to stem base rot resistance identification, and the steps were as follows: Conidium culture: a wild type strain of one of the wheat stem base rot pathogens, Pseudocercosporella herpotrichoides, was selected as the strain, which was cultured on PDA solid medium for activation. A solid with the stem base rot strain was taken from the above-mentioned medium by a disc sampler and placed in 250 g of millet solid medium which was cleaned, boiled, dried and sterilized. The mixture was cultured at 25℃ for 6-7 days (the medium was shaken every day), and wheat stem base rot pathogenic conidium-containing diseased grains were obtained for standby use.

[0045] Diseased grain mixed soil method: before inoculation, the soil culture medium of the test plants was pressed to 1 cm deep at the edge, and then the diseased grains: soil culture medium: water = 1:10:1 (volume ratio) were mixed and placed in the flowerpot to complete the inoculation. The wheat stem base disease situation was investigated and counted 21 days after inoculation, and the stem base rot resistance of the test plants was evaluated according to the disease index.

[0046] The test results are shown in Table 2. Figure 4 Figure 4 Table 2: Resistance phenotype of the test plants to stem base rot fungus Figure 4 Table 2: Resistance phenotype of the test plants to stem base rot fungus TaSYT5 ​The disease index of the gene silencing plants TaSYT5-Ri1-T2, TaSYT5-Ri7-T2 and TaSYT5-Ri8-T2 was significantly reduced compared with the wild type material Fielder. It is shown that TaSYT5 The gene silencing plants have stronger resistance to the stem base rot pathogen. It is shown that TaSYT5 TaSYT5 is an important stem base rot disease-related gene, TaSYT5 TaSYT5 The gene plays a negative regulatory role in the interaction between plants and stem base rot, and inhibiting the expression of the gene can improve the resistance of plants to stem base rot, and inhibiting the expression of the gene can cultivate plant varieties resistant to stem base rot.

Claims

1. TaSYT5 Use of a gene or its related biological material in modulating plant resistance to stem base rot; characterized in that, The TaSYT5 The nucleotide sequence of the gene is shown in SEQ ID No: 1; The related biological material is a nucleic acid molecule containing the TaSYT5 expression cassette of the gene, a recombinant vector, a recombinant microorganism or a transgenic cell line or TaSYT5 the TaSYT5 protein encoded by the gene.

2. Use according to claim 1, characterized in that: The modulating plant resistance to stem base rot is manifested by inhibiting TaSYT5 expression of the gene, increasing the plant resistance to stem base rot.

3. Use according to claim 2, characterized in that, The inhibition TaSYT5 Expression of the gene is achieved by introducing into the plant an RNA interference fragment, an RNA interference vector, a homologous recombination fragment, a homologous recombination vector, or a gene editing tool.

4. Use according to claim 3, characterized in that, The RNA interference vector contains SEQ ID No: 1 from the 5' end 823-1064 nucleotides.

5. Use according to any one of claims 1 to 3, characterized in that, TaSYT5 The protein encoded by the gene is TaSYT5 protein, and the amino acid sequence of the TaSYT5 protein is shown as SEQ ID No:

2.

6. Use according to any one of claims 1 to 3, characterized in that, The plant is a monocotyledon or a dicotyledon, the monocotyledon is a gramineous plant, the gramineous plant is a plant of the family Poaceae, and the plant of the family Poaceae is wheat.

7. A method of increasing resistance of a plant to stem base rot comprising inhibiting expression of a gene in a recipient plant. TaSYT5 gene in a recipient plant.

8. A method for breeding a plant variety that is resistant to stem base rot, comprising inhibiting expression of a gene in a recipient plant to obtain a gene-silenced plant that is resistant to stem base rot. TaSYT5 gene.

9. The method for breeding plant variety resistant to stem base rot according to claim 8, comprising transforming pWMB110-TaSYT5-RNAi silencing vector into the recipient plant by Agrobacterium-mediated transformation, inhibiting the expression of TaSYT5 the gene and obtaining the gene-silenced plant resistant to stem base rot.

10. A method for breeding plant varieties resistant to stem base rot, comprising the following steps: by editing TaSYT5 Genes were used to reduce the expression level and / or activity of the TaSYT5 protein in plants, resulting in plant varieties resistant to stem rot.

Images