Application of wheat disease-resistant factor TaCBL4 in prevention and treatment of basal stem rot

Abstract

The invention belongs to the technical field of genetic engineering, and relates to application of a wheat disease-resistant factor TaCBL4 in prevention and treatment of basal stem rot. A wheat strain capable of overexpressing the wheat disease-resistant factor gene TaCBL4 is obtained by adopting an agrobacterium-mediated method, and the condition that the wheat disease-resistant factor TaCBL4 plays a positive regulation role in the immune response of wheat stem rot resistance is known by comparing the morbidity condition and the fungal biomass condition of a receptor wheat Fielder infected with stem rot pathogens. On the basis, the wheat disease-resistant factor TaCBL4 provided by the invention can be used for creating a stem rot resistant wheat germplasm material, and plays an important role in prevention and treatment of wheat stem 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 TaCBL4 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 that causes root and stem base rot due to the single or combined infection of various pathogens. The main pathogen causing stem base rot is *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 Stem base rot is a soil-borne disease of wheat that not only causes yield loss but also reduces wheat quality, seriously threatening wheat production safety. Discovering wheat resistance genes and improving wheat's disease resistance has become an important means of controlling wheat stem base rot.

[0003] Fusarium graminearum overwinters as mycelium or sporophytes on crop residues. 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 major contributing factors to the increased incidence of stem rot in many regions. As a soil-borne disease, the pathogen infects the wheat roots, leading to disease at the base of the stem and leaf sheaths. For infected seeds or after sowing, Fusarium graminearum inhibits coleoptile growth, causing seed rot and reducing germination rates. Wheat plants infected with the pathogen often develop dark brown streaks or spots at the base of the stem, which spread upwards along the leaf sheaths, nodes, and stem. If temperature and humidity are suitable, a pinkish mold layer will appear between the nodes. At maturity, severely infected wheat suffers from empty or white ears because the vascular bundles are blocked by mycelium, preventing the transport of nutrients and water. Promoting the planting of disease-resistant varieties is the most economical and effective way to control the spread of wheat diseases, and screening for superior resistance 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 more disease-resistant genes, expand methods for wheat resistance to stem rot, and create disease-resistant germplasm materials.

[0004] During their growth and development, plants are exposed to various biotic and abiotic stresses. Increasing research indicates that stress responses depend on complex interactions between multiple signaling pathways within plant cells. These regulatory networks link plant growth and stress tolerance, thereby mitigating the impact of environmental changes on plants. Therefore, understanding the molecular regulatory networks associated with stress responses and plant development is crucial for improving crop disease resistance and yield. Calcium ion signaling is a key signaling molecule in plant immunity; calcium ions are often considered a "second messenger." When plants are infected by microorganisms, they transiently produce calcium ions. 2+ Changes in calcium concentration trigger a series of immune defense responses, including calcium ion signaling and gene expression regulation. In cells, calcium signals bind to specific calcium-sensing proteins. Currently, four major calcium-sensing proteins are known in the plant kingdom: calmodulin (CaM), calmoneurin B protein (CBL), calcium-dependent protein kinase (CDPK), and calcium ion or calmodulin-dependent protein kinase (CCaMK). These calcium-sensing proteins play important regulatory roles in plants, jointly participating in the plant's response to and transmission of calcium signals. These four calcium-sensing proteins affect calcium at the transcriptional or post-transcriptional levels. 2+ The CBL-CIPK signaling system plays a crucial regulatory role in various physiological processes in plants, including but not limited to responses to biotic and abiotic stresses, regulation of growth and development, nutrient absorption and utilization, and regulation of ROS (reactive oxygen species) signaling. Through precise regulation of these physiological processes, the CBL-CIPK signaling system ensures that plants can maintain normal life activities and adapt to various challenges in complex and changing environments. Therefore, the CBL-CIPK signaling mechanism plays an important role in the entire growth and development process of plants. TaCBL4 belongs to the wheat CBL-CIPK signaling system; its disease resistance and applications are relatively limited and require further in-depth research. Summary of the Invention

[0005] To clarify the role of wheat TaCBL4 in the interaction between wheat and pathogens, and to provide more superior disease-resistant genes, this invention provides the application of wheat disease resistance factor TaCBL4 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 TaCBL4 protein described in this invention is represented by "TaCBL4" in non-italicized font. TaCBL4 Genes in italic font TaCBL4 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. TaCBL4 The transcription or translation of genes, or the enhancement of the expression level of TaCBL4 protein in the plant; The TaCBL4 The gene encodes the TaCBL4 protein; The amino acid sequence of the TaCBL4 protein is shown in SEQ ID NO: 1.

[0008] SEQ ID NO: 1 is as follows: MCGAFSSPSPRKREQRAQGYEEPAVLAAETSFTVNEVEALYELYKKLSFSIFKDGLIHKEEFRLALFRTSRGANLFADRVFDLFDLKRNGVIEFGEFVRSLSIFHPKAPES EKTAFAFKLYDLRGTGYIEKEELREMVVALLDESDLCLSDSAVEEIVHNTFSQADSDGDGRIDPKEWEEFVKQNPASLRNMSLPYLQDITTTFPSFVMHSEVEDYSGISK*.

[0009] In SEQ ID NO:1, "*" indicates a terminator.

[0010] Furthermore, in the method for producing plants resistant to stem base rot provided by the present invention, the... TaCBL4 The CDS sequence of the gene is shown in SEQ ID NO: 2.

[0011] SEQ ID NO: 2 is as follows: .

[0012] Furthermore, in the method for producing a plant resistant to stem rot provided by the present invention, the plant is wheat.

[0013] 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 .

[0014] 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. TaCBL4 A gene vector is used to transform Agrobacterium, which is then used to infect the plant.

[0015] 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.

[0016] SEQ ID NO: 3 is as follows: CAGGTCGACTCTAGAGGATCCATGGGCTGCGCGTTCTCGTC.

[0017] SEQ ID NO: 4 is as follows: GAGCTCGGTACCCGGGGATCCTTTGCTGATTCCACTGTAGT.

[0018] On the other hand, this invention relates to... TaCBL4 The application of genes in the prevention and control of stem base rot enhances the plant's... TaCBL4 The transcription or translation of genes, or the enhancement of the expression level of TaCBL4 protein in the plant, can improve the plant's resistance to stem rot. The TaCBL4 The gene encodes the TaCBL4 protein; The amino acid sequence of the TaCBL4 protein is shown in SEQ ID NO: 1.

[0019] Furthermore, in the present invention provided TaCBL4 In the application of genes in the prevention and control of stem base rot, the aforementioned TaCBL4 The CDS sequence of the gene is shown in SEQ ID NO: 2.

[0020] Furthermore, in the present invention provided TaCBL4 The application of genes in the prevention and control of stem base rot, the plant in question is wheat.

[0021] Furthermore, in the invention provided TaCBL4 In the application of gene therapy in the control of stem base rot, the pathogen of which is *Fusarium graminearum* is used. Fusarium pseudograminearum .

[0022] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages: This invention demonstrates the feasibility of using the wheat resistance factor TaCBL4 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 wheat with Fielder recipient varieties after infection with Fusarium pseudogramenosus race WZ-8A, it is determined that the wheat resistance factor TaCBL4 plays a positive regulatory role in the immune response against wheat stem rot. Based on the characteristics of the wheat resistance factor TaCBL4, it can be used to create wheat germplasm materials resistant to stem rot and play a practical role in the control of wheat stem rot. This invention provides a preferred method: constructing a recombinant overexpression vector containing the gene encoding the wheat resistance factor TaCBL4, 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 rot and a method for controlling wheat stem rot, thereby reducing pathogen resistance and environmental pollution caused by pesticide overuse. Attached Figure Description

[0023] 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.

[0024] Figure 1 For wheat TaCBL4 A schematic diagram of a gene overexpression recombinant vector.

[0025] Figure 2 For overexpression TaCBL4 Image showing the results of obtaining transgenic wheat plants from the gene. A represents the T3 generation. TaCBL4 -OE2、 TaCBL4 -OE5、 TaCBL4 Molecular detection of -OE6; B is TaCBL4 Gene expression in the control group (Fielder) and overexpression lines ( TaCBL4 -OE2、 TaCBL4 -OE5、 TaCBL4 The relative expression levels of -OE6; C represents the control group (Fielder) and the overexpression line ( TaCBL4 -OE2、 TaCBL4 -OE5、 TaCBL4 The pre-inoculation phenotype of -OE6.

[0026] Figure 3 For overexpression TaCBL4 A diagram illustrating the disease resistance verification of transgenic wheat plants. Specifically, it shows...TaCBL4 Gene overexpression lines ( TaCBL4 -OE2、 TaCBL4 -OE5、 TaCBL4 Disease incidence and fungal DNA / wheat DNA biomass (fungal biomass) statistics of the OE6 and control (Fielder) plants. Detailed Implementation

[0027] 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.

[0028] Example 1 This embodiment provides the method for obtaining the gene encoding the wheat disease resistance factor TaCBL4.

[0029] The nucleotide sequence of the gene encoding the wheat disease resistance factor TaCBL4 was obtained from the Ensembl Plants website (https: / / plants.ensembl.org / Triticum_aestivum / Info / Index). An amplification design was then developed based on this nucleotide sequence. TaCBL4 Primers for the full-length gene, the primers used are: TaCBL4 -F: 5'-ATGGGCTGCGCGTTCTCGTCGTCGC-3'; TaCBL4 -R:5'-TTATTTGCTGATTCCACTGTAGTCT-3'.

[0030] Using cDNA from Fielder wheat plants as a template, amplification TaCBL4 Genes were sequenced to obtain... TaCBL4 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.

[0031] Example 2 This embodiment provides the construction of transgenic lines (overexpression lines) and the identification of their disease resistance.

[0032] According to those skilled in the art and in conjunction with existing technology, utilizing the amplification in Example 1... TaCBL4 The full-length gene sequence (SEQ ID NO: 2) was obtained through a gateway reaction of homologous recombination. TaCBL4The full-length amplified gene fragment was constructed into the overexpression vector CUB to form a recombinant overexpression vector. TaCBL4 -CUB. The successfully constructed recombinant overexpression vector... TaCBL4 -CUB was transfected into Fielder wheat plants via Agrobacterium (EHA105)-mediated transfection to obtain plants capable of overexpressing CUB. TaCBL4 Genetically modified plants.

[0033] Constructing recombinant overexpression vectors TaCBL4 -CUB, the primers used are: TaCBL4 -CUB-F: 5'-CAGGTCGACTCTAGAGGATCCATGGGCTGCGCGTTCTCGTC-3'; TaCBL4- CUB-R: 5'-GAGCTCGGTACCCGGGGATCCTTTGCTGATTCCACTGTAGT-3'.

[0034] Transgenic plants were subjected to PCR and qRT-PCR detection. Based on the detection results, positive lines (OE2, OE5, and OE6) were selected, and stable overexpression lines were obtained through multiple generations of culture. TaCBL4 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. TaCBL4 Resistance of genetically modified strains.

[0035] The T3 generation, built based on the above technical approach TaCBL4 Gene overexpression lines ( TaCBL4 -OE2、 TaCBL4 -OE5、 TaCBL4 Wheat plants in the control group (Fielder) and the control group (OE6) were inoculated with Fusarium graminearum physiological race 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.

[0036] TaCBL4 Gene overexpression lines ( TaCBL4 -OE2、 TaCBL4 -OE5、 TaCBL4 Disease incidence in plants of type OE6 and control group (Fielder) is as follows: Figure 3 As shown. By Figure 3 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, TaCBL4The severity of stem rot in the gene-overexpressing lines was lower than that in the control group.

[0037] Example 3 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.

[0038] T3 generation cells that tested positive for PCR TaCBL4 Gene overexpression lines ( TaCBL4 -OE2、 TaCBL4 -OE5、 TaCBL4 Both the control group (Fielder) and the control group (-OE6) were inoculated with Fusarium pseudobulb physiological race WZ-8A after the second leaf unfolded, and samples were taken for analysis 16 days after inoculation.

[0039] 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 synthesized using the SMART method. qRT-PCR was then used for detection. TaCBL4 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. 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.

[0040] The qRT-PCR primer sequences are: Ta18SrRNA -F:5'-GTGACGGGTGACGGAGAATT -3'; Ta18SrRNA -R:5'-GACACTAATGCGCCCGGTAT-3'.

[0041] FpGAPDH -F:5'-GAAGGTCATCATCTCTGCCC-3'; FpGAPDH -R: 5'-GTCTTCTGGGTGGCAGTGTA-3'.

[0042] 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.

[0043] Figure 3 The left-middle figure is provided by an embodiment of the present invention. TaCBL4 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 3 As can be seen, compared with the control group Fielder plants, TaCBL4 The fungal biomass of the overexpressing transgenic plants was significantly reduced.

[0044] 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 for obtaining plants resistant to stem base rot, characterized in that, include: Enhance the plant TaCBL4 The transcription or translation of genes, or the enhancement of the expression level of TaCBL4 protein in the plant; The TaCBL4 The gene encodes the TaCBL4 protein; The amino acid sequence of the TaCBL4 protein is shown in SEQ ID NO:

1.

2. The method for obtaining plants resistant to stem base rot according to claim 1, characterized in that, The TaCBL4 The CDS sequence of the gene is shown in SEQ ID NO:

2.

3. The method for obtaining plants resistant to stem base rot according to claim 1, characterized in that, The plant in question is wheat.

4. The method for obtaining plants resistant to stem base rot according to claim 1, characterized in that, The pathogen causing the stem base rot is *Fusarium graminearum*. Fusarium pseudograminearum .

5. The method for obtaining plants resistant to stem base rot according to claim 1, characterized in that, Construct overexpression TaCBL4 A gene vector is used to transform Agrobacterium, which is then used to infect the plant.

6. The method for obtaining plants resistant to stem base rot according to claim 5, characterized in that, 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.

7. TaCBL4 The application of genes in the prevention and control of stem base rot is characterized by, Enhance the plant TaCBL4 The transcription or translation of genes, or the enhancement of the expression level of TaCBL4 protein in the plant, can improve the plant's resistance to stem rot. The TaCBL4 The gene encodes the TaCBL4 protein; The amino acid sequence of the TaCBL4 protein is shown in SEQ ID NO:

1.

8. The method according to claim 7 TaCBL4 The application of genes in the prevention and control of stem base rot is characterized by, The TaCBL4 The CDS sequence of the gene is shown in SEQ ID NO:

2.

9. The method according to claim 7 TaCBL4 The application of genes in the prevention and control of stem base rot is characterized by, The plant in question is wheat.

10. The claim 7 TaCBL4 The application of genes in the prevention and control of stem base rot is characterized by, The pathogen causing the stem base rot is *Fusarium graminearum*. Fusarium pseudograminearum .

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