Application of wheat disease resistance factor TaCIPK10 in the prevention and treatment of stem base rot

Abstract

The application belongs to the technical field of genetic engineering, and relates to application of a wheat disease resistance factor TaCIPK10 in prevention and treatment of stem base rot. TaCIPK10 The application obtains a wheat strain capable of overexpressing a wheat disease resistance factor gene TaCIPK10 by using an agrobacterium-mediated method, and obtains the wheat disease resistance factor TaCIPK10 by comparing the disease incidence and fungal biomass after infection of a stem base rot pathogen in the wheat Fielder, and finds that the wheat disease resistance factor TaCIPK10 plays a positive regulation role in the immune response of the wheat against the stem base rot. Based on this, the wheat disease resistance factor TaCIPK10 can be used to create a wheat germplasm material resistant to the stem base rot, and plays an important role in prevention and treatment of the wheat stem base rot.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the application of wheat disease resistance factor TaCIPK10 in the prevention and control of stem base rot. Background Technology

[0002] Stem base rot ( Fusarium Crown rot (FCR) is a fungal soil-borne disease, belonging to a class of diseases caused by single or combined infections of various pathogens, resulting in root and stem base rot. The main pathogens include *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 Wheat stem rot is a fungal soil-borne disease that mainly affects the leaf sheaths and stems of the 1st to 2nd nodes at the base of the wheat plant, causing lodging or whiteheads. It generally reduces yield by 5% to 10%, but in severe cases, it can reduce yield by more than 50% or even result in total crop failure.

[0003] *Fusarium graminearum* is the main pathogen causing wheat stem rot. After germination, it forms asexual macroconidia that infect wheat. *Fusarium graminearum* can survive in crop residues for up to three years. No-till farming and stubble retention to conserve soil moisture and nutrients are the main reasons for the increased incidence of stem rot in many regions. After wheat is infected with the pathogen, the leaf sheaths at the base of the stem change color from light brown to dark brown as the disease progresses, eventually rotting, making the plant easy to pull out. In high humidity microclimates, a pink or white mold layer appears at the base of the stem. In later stages, severely infected wheat dies, forming white ears, significantly affecting the number of grains per ear and the thousand-grain weight, leading to yield reduction.

[0004] Promoting the planting of disease-resistant varieties is the most economical and effective way to control wheat disease outbreaks, and screening for superior disease-resistant genes is the foundation for creating disease-resistant varieties. However, because wheat resistance to stem rot is a complex quantitative trait with a complex genetic basis and is easily affected by the environment, there are certain difficulties in screening and utilizing resistance genes. Currently available germplasm resources and resistance genes are extremely scarce, and there is an urgent need to discover new disease-resistant genes, expand methods for wheat resistance to stem rot, and create new disease-resistant germplasm materials. The wheat disease resistance factor TaCIPK10 belongs to the CBL-CIPK signaling system, but its application in the control of stem rot has not been observed. Summary of the Invention

[0005] To clarify the role of wheat TaCIPK10 in the interaction between wheat and pathogens, and to provide more superior resistance genes, this invention provides the application of wheat disease resistance factor TaCIPK10 in the control of stem base rot.

[0006] To ensure a complete and unambiguous understanding of the technical solution of this invention, it should be noted that the TaCIPK10 protein described in this invention is represented by "TaCIPK10" in non-italicized font. TaCIPK10 Genes in italic font TaCIPK10 This indicates that, of course, those skilled in the art can clearly and completely understand the meaning and description of the relevant genes and their encoded proteins based on the description in this invention.

[0007] On one hand, the present invention relates to a method for obtaining plants resistant to stem base rot, comprising: increasing the concentration of certain substances in the plant. TaCIPK10 The transcription or translation of genes, or the enhancement of the expression level of TaCIPK10 protein in the plant;

[0008] The TaCIPK10 The gene encodes the TaCIPK10 protein;

[0009] The amino acid sequence of the TaCIPK10 protein is shown in SEQ ID NO: 1.

[0010] SEQ ID NO: 1 is as follows:

[0011] MVEKKGNILMERYEMGRRLLGQGSFAKVYYGRSLKTSQSVAIKVIDKEKIFKCGLMDQVRREISVMKLVKHPNIVQLYEVMATKTKIFFVLEYVKGGELFNKVQRGRLKED VARKYFQQLNSAVDFCHSRGVYHRDLKPENLLLDENRNLKISDFGLSALAECKRQDGLLHTTCGTPAYVAPELISKKGYDGAKADIWACGVILYVLLAGYLPFQDKNLMD MYKKIYKAELKWPSWFSSDARRLLRRILDPNPGTRISFSEILDNSWFRTGLDKGLISYNTPTEGAVDMDPTCDPFSSCTTETIQEATELTNLNAFDIISLSSGFDLSG MFEDKSNKESKFTSTNTAATIITKLEDIAKRLRLRLMKRDGGLLKMQSLQPGRKGVMSIDTEIFQIAPNFHLVEIRKTNGDTLEYQKVKHDMRPALKDIVWAWQGEQP*.

[0012] In SEQ ID NO:1, "*" indicates a terminator.

[0013] Furthermore, in the method for producing plants resistant to stem base rot provided by the present invention, the... TaCIPK10 The CDS sequence of the gene is shown in SEQ ID NO: 2.

[0014] SEQ ID NO: 2 is as follows:

[0015]

[0016] Furthermore, in the method for producing a plant resistant to stem rot provided by the present invention, the plant is wheat.

[0017] Furthermore, in the method for producing plants resistant to stem base rot provided by the present invention, the pathogen causing the stem base rot is *Fusarium graminearum*. Fusarium pseudograminearum .

[0018] Furthermore, in the method for producing plants resistant to stem base rot provided by this invention, an expression of the aforementioned [organization / product] is constructed. TaCIPK10 A gene vector is used to transform Agrobacterium, which is then used to infect the plant.

[0019] Furthermore, in the method for constructing plants resistant to stem base rot provided by the present invention, the upstream primer for constructing the vector is shown in SEQ ID NO: 3, and the downstream primer is shown in SEQ ID NO: 4.

[0020] SEQ ID NO: 3 is as follows:

[0021] CAGGTCGACTCTAGAGGATCCATGGTAGAGAAGAAGGGAAA.

[0022] SEQ ID NO: 4 is as follows:

[0023] GAGCTCGGTACCCGGGGATCCGGGCTGCTCACCTTGCCAAG.

[0024] On the other hand, this invention relates to... TaCIPK10 The application of genes in the prevention and control of stem base rot enhances the plant's... TaCIPK10 The transcription or translation of genes, or the enhancement of the expression level of TaCIPK10 protein in the plant, can improve the plant's resistance to stem rot.

[0025] The TaCIPK10 The gene encodes the TaCIPK10 protein;

[0026] The amino acid sequence of the TaCIPK10 protein is shown in SEQ ID NO: 1.

[0027] Furthermore, in the present invention provided TaCIPK10 In the application of genes in the prevention and control of stem base rot, the aforementioned TaCIPK10 The CDS sequence of the gene is shown in SEQ ID NO: 2.

[0028] Furthermore, in the present invention provided TaCIPK10 The application of genes in the prevention and control of stem base rot, the plant in question is wheat.

[0029] Furthermore, in the present invention provided TaCIPK10 In the application of gene therapy in the control of stem base rot, the pathogen of which is *Fusarium graminearum* is used. Fusarium pseudograminearum .

[0030] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0031] This invention demonstrates the feasibility of using the wheat resistance factor TaCIPK10 to breed wheat varieties resistant to stem rot. Using Agrobacterium-mediated transformation, this invention obtains wheat lines capable of overexpressing this resistance factor. By comparing the disease incidence and fungal biomass of TaCIPK10 with that of ordinary Fielder wheat after infection with Fusarium graminearum race WZ-8A, it is determined that TaCIPK10 plays a positive regulatory role in the immune response against wheat stem rot. Based on the characteristics of TaCIPK10, it can be used to create wheat germplasm materials resistant to stem rot and play a practical role in the prevention and control of wheat stem rot. This invention provides a preferred method: constructing a recombinant overexpression vector containing the gene encoding the wheat resistance factor TaCIPK10, then transferring it into wheat plants, and obtaining stably inherited wheat lines capable of overexpressing this resistance factor through multiple generations of culture. This invention provides a technical approach for breeding wheat varieties resistant to stem base rot, and also offers a new method for controlling wheat stem base rot, thereby reducing pathogen resistance and environmental pollution caused by pesticide overuse. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 for TaCIPK10 A schematic diagram of a gene overexpression recombinant vector.

[0034] Figure 2 For T3 generation TaCIPK10 -OE1、 TaCIPK10 -OE2、 TaCIPK10 The molecular detection results of -OE5 are shown in the figure.

[0035] Figure 3 for TaCIPK10 Gene expression in control (Fielder) plants and transgenic lines (overexpression lines) TaCIPK10 -OE1、 TaCIPK10 -OE2、 TaCIPK10 A statistical chart of the relative expression levels of -OE5.

[0036] Figure 4 for TaCIPK10 Gene overexpression lines ( TaCIPK10 -OE1、 TaCIPK10 -OE2、 TaCIPK10 Disease incidence and pathogen DNA / wheat DNA biomass statistics in plants of type OE5 and control group (Fielder). Detailed Implementation

[0037] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental and detection methods described in each embodiment are conventional methods; the reagents and materials described are commercially available unless otherwise specified. Unless otherwise specified, all percentages in the following embodiments refer to mass percentages. Unless otherwise specified, all proportions in the following embodiments refer to mass ratios.

[0038] Example 1

[0039] This embodiment provides the method for obtaining the gene encoding the wheat disease resistance factor TaCIPK10.

[0040] The nucleotide sequence of the gene encoding the wheat disease resistance factor TaCIPK10 was obtained from the Ensembl Plants website (https: / / plants.ensembl.org / Triticum_aestivum / Info / Index). Primers for amplifying the full length of the TaCIPK10 gene were designed based on this nucleotide sequence. The primers used are:

[0041] TaCIPK10 -F: 5'-ATGGTAGAGAAGAAGGGAAATATCT-3';

[0042] TaCIPK10 -R: 5'-CTAGGGCTGCTCACCTTGCCAAGCC-3'.

[0043] Using cDNA from Fielder wheat plants as a template, amplification TaCIPK10 Genes were sequenced to obtain... TaCIPK10 The CDS sequence of the gene is shown in SEQ ID NO: 2, and the amino acid sequence it encodes is shown in SEQ ID NO: 1.

[0044] Example 2

[0045] This embodiment provides the construction of transgenic lines (overexpression lines) and the identification of their disease resistance.

[0046] According to those skilled in the art and in conjunction with existing technology, utilizing the amplification in Example 1... TaCIPK10 The full-length gene sequence, through the gateway reaction of homologous recombination, will TaCIPK10 The full-length amplified gene fragment was constructed into the overexpression vector CUB to form a recombinant overexpression vector. TaCIPK10 -CUB. The successfully constructed recombinant overexpression vector... TaCIPK10 -CUB was transfected into Fielder wheat plants via Agrobacterium (EHA105)-mediated transfection to obtain plants capable of overexpressing CUB. TaCIPK10 Genetically modified plants.

[0047] Construction of recombinant overexpression vectors TaCIPK10 -CUB, the primers used are:

[0048] TaCIPK10 -CUB-F: 5'-CAGGTCGACTCTAGAGGATCCATGGTAGAGAAGAAGGGAAA-3';

[0049] TaCIPK10- CUB-R: 5'-GAGCTCGGTACCCGGGGATCCGGGCTGCTCACCTTGCCAAG-3'.

[0050] Transgenic plants were subjected to PCR and qRT-PCR detection. Based on the detection results, T1 generation positive lines (OE1, OE2, and OE5) were selected, and stable overexpression lines were obtained through multiple generations of culture. TaCIPK10 High-generation wheat lines of the gene were inoculated with *Fusarium graminearum* physiological race WZ-8A, using Fielder wheat plants as a control, to determine overexpression. TaCIPK10 Resistance of genetically modified strains.

[0051] The T3 generation, built based on the above technical approach TaCIPK10 Gene overexpression lines ( TaCIPK10 -OE1、 TaCIPK10 -OE2、 TaCIPK10 Wheat plants in the control group (Fielder) and the control group (OE5) were inoculated with Fusarium graminearum WZ-8A after the second leaf unfolded, and the disease incidence of each group was observed 16 days after inoculation. The inoculation method and resistance evaluation were based on the local standard DB41 / T 2392-2023 Technical Specification for Evaluation of Wheat Resistance to Stem Base Rot.

[0052] TaCIPK10 Gene overexpression lines ( TaCIPK10 -OE1、 TaCIPK10 -OE2、 TaCIPK10Disease incidence in plants of type OE5 and control group (Fielder) is as follows: Figure 4 As shown. By Figure 4 It was found that brown spots were observed at the base of wheat stems in all groups under infection with Fusarium pseudogramensis physiological race WZ-8A. However, TaCIPK10 The severity of stem rot in the gene-overexpressing lines was lower than that in the control group.

[0053] Example 3

[0054] This embodiment provides an analysis of the biomass ratio of pathogen DNA to wheat DNA after transgenic lines were inoculated with Fusarium pseudograss physiological race WZ-8A.

[0055] T3 generation cells that tested positive for PCR TaCIPK10 Gene overexpression lines ( TaCIPK10 -OE1、 TaCIPK10 -OE2、 TaCIPK10 Both the control group (Fielder) and the control group (-OE5) were inoculated with Fusarium graminearum WZ-8A after the second leaf unfolded, and samples were taken for analysis 16 days after inoculation.

[0056] Wheat plant stems, approximately 2 cm long from the base, were cut, wrapped in aluminum foil, and flash-frozen in liquid nitrogen at -80˚C for later use. Total RNA was extracted from wheat leaves using the Trizol (TianGen) method, and first-strand cDNA was synthesized using reverse transcriptase XL (AMV). cDNA was then synthesized using the SMART method. qRT-PCR was then used for detection. TaCIPK10 Gene expression levels. Wheat tissue DNA was diluted to 4... 2 4 3 4 4 4 5 4 6 4 7 The DNA of *Fusarium graminearum* race WZ-8A was diluted by the same factor as above. Using the diluted DNA as a template, the wheat ribosomal small subunit gene was used. Ta18SrRNA and the gene of glyceraldehyde-3-phosphate dehydrogenase in pathogens FpGAPDH Primers were used to perform qRT-PCR, and a standard curve was plotted between the obtained Cq values ​​and the dilution gradient.

[0057] The DNA from wheat tissues inoculated with Fusarium pseudograecum WZ-8A was diluted to 4... 4 Using wheat as a template, Ta18SrRNA and germs FpGAPDH Using primers, qRT-PCR was performed. The obtained Cq values ​​were substituted into the standard curve obtained above to calculate the ratio of pathogen DNA to wheat DNA biomass.

[0058] The qRT-PCR primer sequences are:

[0059] Ta18SrRNA -F:5'-GTGACGGGTGACGGAGAATT -3';

[0060] Ta18SrRNA -R:5'-GACACTAATGCGCCCGGTAT-3'.

[0061] FpGAPDH -F:5'-GAAGGTCATCATCTCTGCCC-3';

[0062] FpGAPDH -R: 5'-GTCTTCTGGGTGGCAGTGTA-3'.

[0063] Before use, the specificity and amplification efficiency of the primers for quantitative PCR must be tested, and the amplification efficiency should be ≥90%. Using an AceQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and a Bio-Rad CFXManager quantitative PCR instrument (Bio-rad, Hercules, California), qRT-PCR was performed using DNA from each gene sampling site as a template, following the instruction manual. Three replicates were performed for each gene. The Ct values, mean, and standard deviation of each replicate were generated by the quantitative PCR instrument by manually adjusting the baseline. The Delta Ct method was used to analyze the experimental data and determine the biomass ratio of pathogen DNA to wheat DNA.

[0064] Figure 4 The left-middle figure is provided by an embodiment of the present invention. TaCIPK10 A schematic diagram showing the resistance of overexpressing transgenic plants inoculated with Fusarium graminearum. The right figure shows the biomass analysis 16 days after inoculation. Figure 4 As can be seen, compared with the control group Fielder plants, TaCIPK10 The fungal biomass of the overexpressing transgenic plants was significantly reduced.

[0065] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate preferred embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A method of obtaining a plant resistant to stem base rot, characterized by, Comprising: overexpressing a gene in said plant TaCIPK10 TaCIPK10 protein in said plant; The TaCIPK10 gene encodes the TaCIPK10 protein; The amino acid sequence of the TaCIPK10 protein is shown as SEQ ID NO: 1; The plant is wheat; The pathogenic fungus of the stem base rot is Pseudocercosporella herpotrichoides Fusarium pseudograminearum .

2. The method of obtaining a plant resistant to stem base rot according to claim 1, characterized in that, The TaCIPK10 The CDS sequence of the gene is shown as SEQ ID NO:

2.

3. The method of obtaining a plant resistant to stem base rot according to claim 1, characterized in that, Constructing a vector overexpressing the gene, transforming the vector into Agrobacterium, and infecting the plant with the Agrobacterium. TaCIPK10 Constructing a vector overexpressing the gene, transforming the vector into Agrobacterium, and infecting the plant with the Agrobacterium.

4. The method of obtaining a plant resistant to stem base rot according to claim 3, characterized by, The upstream primer for constructing the vector is shown as SEQ ID NO: 3, and the downstream primer is shown as SEQ ID NO:

4.

5. TaCIPK10 The use of the gene in the prevention and treatment of basal stem rot, characterized in that, Overexpression in plants TaCIPK10 Genes, or overexpression of the TaCIPK10 protein in plants; The TaCIPK10 gene encodes the TaCIPK10 protein; The amino acid sequence of the TaCIPK10 protein is shown as SEQ ID NO: 1; The plant is wheat; The pathogenic fungus of the stem base rot is Pseudocercosporella herpotrichoides Fusarium pseudograminearum .

6. The method according to claim 5 TaCIPK10 The application of genes in the prevention and control of stem base rot is characterized by, The TaCIPK10 The CDS sequence of the gene is shown as SEQ ID NO: 2.

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