Application of wheat disease resistance factor TaPKS26 in leaf rust prevention

Abstract

The present application belongs to the technical field of agricultural bioengineering, and relates to application of a wheat disease resistance factor TaPKS26 in leaf rust prevention and treatment. The CDS sequence of the coding gene of the wheat disease resistance factor TaPKS26 is shown as SEQ ID NO:1, and the present application adopts an agrobacterium-mediated method to obtain a transgenic wheat strain overexpressing the gene TaPKS26. After being infected with the physiological race THT and PHT of leaf rust, the transgenic wheat strain overexpressing the gene TaPKS26 is compared with Fielder wheat, and the results show that the resistance of the transgenic wheat strain to wheat leaf rust is enhanced, and the expression amount of a disease resistance related marker gene is up-regulated, indicating that the wheat disease resistance factor TaPKS26 plays a positive regulation role in the defense response of wheat against leaf rust. The present application provides a new gene resource and technical idea for breeding of a leaf rust disease resistance variety.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bioengineering technology, specifically relating to the application of wheat disease resistance factor TaPKS26 in the control of leaf rust. Background Technology

[0002] Wheat leaf rust is another important rust disease in my country after stripe rust. The pathogen is *Puccinia triticina*, a typical biotrophic metaphytic rust fungus that relies on a living host for nutrients. Its life cycle includes asexual and sexual stages. The fungus primarily infects wheat leaves, occasionally leaf sheaths and stems. After infection, brick-red uredinia are scattered on the leaves, affecting the photosynthetic rate and consequently grain filling, leading to reduced yield and, in severe cases, leaf death. Leaf rust is mainly spread by air currents. With global warming, meteorological conditions are becoming more favorable for wheat leaf rust, potentially posing a greater threat to wheat production in the future.

[0003] Currently, the main control measures for wheat leaf rust include breeding resistant varieties and chemical control. However, the frequent use of triazole fungicides has led to the emergence of resistant strains. On the other hand, frequent variations in the virulence of the original fungus have resulted in the continuous evolution of races, and existing wheat varieties lack resistant resources for wheat leaf rust. Therefore, it is urgent to explore new wheat leaf rust resistance gene resources, identify functional genes with application potential, and create new resistant germplasm materials to achieve green and sustainable control of the disease. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention constructs an overexpression vector for the wheat disease resistance factor TaPKS26 encoding gene using genetic engineering technology. Transgenic plants overexpressing TaPKS26 are obtained using Agrobacterium-mediated transformation. Further research into the disease resistance of these transgenic plants reveals that TaPKS26 plays a positive regulatory role in the interaction between wheat and leaf rust pathogens, and that transgenic plants overexpressing TaPKS26 exhibit enhanced resistance to leaf rust.

[0005] To ensure a complete and unambiguous understanding of the technical solution of this invention, it should be noted that the wheat disease resistance factor encoding gene TaPKS26 is represented by italicized "TaPKS26", while the wheat disease resistance factor protein TaPKS26 is represented by non-italicized "TaPKS26". 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 of this invention. On one hand, this invention provides the application of the wheat disease resistance factor TaPKS26 in the control of leaf rust. The wheat disease resistance factor TaPKS26 plays a positive regulatory role in the interaction between wheat and the leaf rust pathogen. The CDS sequence of the wheat disease resistance factor encoding gene is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0006] Furthermore, in the aforementioned application, overexpression of the wheat disease resistance factor TaPKS26 in wheat enhances wheat's resistance to leaf rust.

[0007] In another aspect, the present invention also provides a method for enhancing wheat resistance to leaf rust, the method comprising overexpressing the wheat resistance factor TaPKS26 in wheat plants.

[0008] Furthermore, the method includes constructing a recombinant overexpression vector and transforming the recombinant overexpression vector into wheat, wherein the recombinant overexpression vector contains the encoding gene of the wheat disease resistance factor TaPKS26.

[0009] Finally, the present invention also provides a method for breeding wheat varieties resistant to leaf rust, which includes overexpressing the wheat disease resistance factor TaPKS26 in wheat to obtain TaPKS26 overexpressing lines.

[0010] Furthermore, in the cultivation method, when the TaPKS26 overexpressing plants are infected with wheat leaf rust pathogen, the disease resistance marker genes TaPR1, TaPR2 and TaPR5 are all upregulated in the overexpressing plants.

[0011] Furthermore, the cultivation method includes the step of constructing an overexpression recombinant vector containing the wheat disease resistance factor gene TaPKS26 and transferring it into wheat plants.

[0012] Furthermore, in the cultivation method, wheat genetic transformation is carried out using Agrobacterium-mediated transformation.

[0013] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0014] (1) Compared with traditional disease-resistant breeding techniques, plant disease-resistant genetic engineering technology can overcome species reproductive isolation and incompatibility in distant hybridization, and can directionally improve target traits in a shorter period of time, providing crops with more comprehensive, continuous, and broad-spectrum disease resistance protection. Through gene function research, this invention has found that the wheat disease resistance factor TaPKS26 plays a positive regulatory role in wheat's defense response to leaf rust, and overexpression of TaPKS26 can enhance wheat's resistance to leaf rust.

[0015] (2) This invention provides a method for breeding wheat varieties resistant to leaf rust. Using genetic engineering technology, the TaPKS26 gene is overexpressed in wheat plants to enhance their resistance to leaf rust pathogens. Experiments show that the TaPKS26-overexpressing wheat obtained by this method exhibits resistance to both THT and PHT of the leaf rust fungus. This invention enriches the leaf rust resistance gene resource library, provides a technical pathway for breeding wheat varieties resistant to leaf rust, and offers an economical and effective new method for the green control of wheat leaf rust. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the overexpression vector TapKS26-CUB.

[0017] Figure 2 Figure 1 shows the identification results of the positive TaPKS26 overexpressing lines TaPKS26-OE5-T3, TaPKS26-OE8-T3, and TaPKS26-OE9-T3. Figure A shows the PCR detection results; Figure B shows the relative expression levels of TaPKS26 in TaPKS26-OE5-T3, TaPKS26-OE8-T3, TaPKS26-OE9-T3, and Fielder plants; Figure C shows the growth and development of TaPKS26-OE5-T3, TaPKS26-OE8-T3, TaPKS26-OE9-T3, and Fielder plants at the seedling stage. "*" indicates significance at the p < 0.05 level.

[0018] Figure 3Phenotypic diagrams and statistical results of the relative expression levels of disease resistance-related marker genes of T3 generation TaPKS26 overexpression lines TaPKS26-OE5, TaPKS26-OE8, TaPKS26-OE9 and Fielder plants after inoculation with leaf rust fungus THT and PHT. Figure A shows the phenotypic results of T3 generation TaPKS26 overexpression lines TaPKS26-OE5, TaPKS26-OE8, TaPKS26-OE9, and Fielder after inoculation with leaf rust fungi THT and PHT; Figure B shows the statistical results of the relative expression level of TaPR1 in T3 generation TaPKS26 overexpression lines TaPKS26-OE5, TaPKS26-OE8, TaPKS26-OE9, and Fielder; Figure C shows the statistical results of the relative expression level of TaPR2 in T3 generation TaPKS26 overexpression lines TaPKS26-OE5, TaPKS26-OE8, TaPKS26-OE9, and Fielder; Figure D shows the statistical results of the relative expression level of TaPR5 in T3 generation TaPKS26 overexpression lines TaPKS26-OE5, TaPKS26-OE8, TaPKS26-OE9, and Fielder. "*" indicates significance at the p<0.05 level. Detailed Implementation

[0019] 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.

[0020] 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.

[0021] 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.

[0022] Example 1

[0023] This embodiment provides the method for obtaining the wheat disease resistance factor gene TapKS26.

[0024] The nucleotide sequence of the wheat disease resistance factor TaPKS26 was obtained from the Ensembl Plants website (https: / / plants.ensembl.org / Triticum_aestivum / Info / Index). The nucleotide sequence of the gene encoding the wheat disease resistance factor is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2. The nucleotide sequence of the wheat disease resistance factor TaPKS26 is as follows:

[0025]

[0026] The amino acid sequence of the wheat disease resistance factor TaPKS26 is as follows:

[0027] MAAVKLEEVRRAQRAEGLATVLAIGTAVPANCVYQATYPDYYFRVTKSEHLPDLKEKFERMCEKSTIRKRHMHLTEEILKKNPSICSHMEPSLDTRHDIVVVEVPKLGKEAAERAIKEWGQPLSKITHVIFCTTSGVDMPGADYQLTRLLGLSPAVKRLMMYQQGCFGGATVLRMAKDIAENNRGARVLVVCSEITAM AFRGPSKSHLDSLVGHALFGDGAAAAIIGADPDEPFEKPLFQLVSASQTILPDSDGAINGHLTEAGLTIHLLKDVPGLISENIEKALEDAFKPLGIHDWNSIFWIAHPGGPAILDMVEEKVGLDKERMRASREVLSEYGNMSSACVLFVLDVMRKTSSQDGHATTGEGKEWGVLFGFGPGLTVETLVLYSVPITATN.

[0028] Primers TaPKS26-F and TaPKS26-R were designed to amplify the TaPKS26 gene based on the nucleotide sequence shown in SEQ ID NO:1. The sequence information of primers TaPKS26-F and TaPKS26-R is detailed in Table 1. Using cDNA from Fielder wheat plants as a template, the TaPKS26 gene was amplified and sequenced. The CDS sequence of the TaPKS26 gene is shown in SEQ ID NO:1, and its encoded amino acid sequence is shown in SEQ ID NO:2.

[0029] Table 1 Primer sequence list

[0030]

[0031]

[0032] Example 2

[0033] This example demonstrates the construction of a wheat disease resistance gene TaPKS26 overexpression line.

[0034] Using the wheat disease resistance gene TaPKS26 amplified in Example 1, the TaPKS26 gene was constructed into the overexpression vector CUB via homologous recombination Gateway technology, obtaining the recombinant overexpression vector TaPKS26-CUB. The successfully constructed overexpression vector TaPKS26-CUB was transfected into wheat Fielder plants using Agrobacterium-mediated transformation to obtain transgenic wheat capable of stably overexpressing the TaPKS26 gene, namely, T0 generation TaPKS26 overexpression plants.

[0035] The recombinant overexpression vector TaPKS26-CUB was constructed using primers TaPKS26-CUB-F and TaPKS26-CUB-R. Primer information for TaPKS26-CUB-F and TaPKS26-CUB-R is shown in Table 1. PCR detection was performed on the obtained T0 generation TaPKS26 overexpressing plants. When the positive lines were further subcultured to the T3 generation, PCR detection was performed on the obtained T3 generation TaPKS26 overexpressing plants using the detection primer pairs TaPKS26-CUB-JC-F and TaPKS26-CUB-JC-R. Primer information is shown in Table 1. The detection fragment is shown in Table 1. Figure 1 As shown.

[0036] Plants overexpressing TaPKS26 that showed a detectable 274bp insertion fragment were identified as positive TaPKS26 overexpression plants. T3 generation positive lines (TaPKS26-OE5-T0, TaPKS26-OE8-T0, and TaPKS26-OE9-T0) were selected to obtain stably heritable high-generation wheat lines overexpressing the TaPKS26 gene: TaPKS26-OE5-T3, TaPKS26-OE8-T3, and TaPKS26-OE9-T3. The disease resistance of these high-generation wheat lines was tested, and the results are as follows: Figure 2 As shown, Figure 2 In the diagram, lane A represents the PCR detection result, lane H2O represents water, lane Fielder represents Fielder wheat, and lanes TaPKS26-OE5-T3, TaPKS26-OE8-T3, and TaPKS26-OE9-T3 represent 10 randomly selected TaPKS26-OE5-T3, 10 TaPKS26-OE8-T3, and 10 TaPKS26-OE9-T3 plants, respectively. The results indicate that all tested plants were positive for TaPKS26 overexpression. Figure 2Figure B shows the relative expression levels of TaPKS26 in TaPKS26-overexpressing plants and Fielder plants. The results showed that the TaPKS26 gene expression levels in T3 generation plants (TaPKS26-OE5-T3, TaPKS26-OE8-T3, and TaPKS26-OE9-T3) were significantly increased compared to Fielder plants. The growth of T3 generation TaPKS26-overexpressing plants and Fielder seedlings was observed and photographed, and the results are shown below. Figure 2 As shown in Figure C, the T3 generation plants overexpressing the TaPKS26 gene and the Fielder plants showed consistent growth and were both growing well.

[0037] Example 3

[0038] This example is for identifying the disease resistance of lines overexpressing the TaPKS26 gene.

[0039] Using T3 generation TaPKS26 overexpression lines (TaPKS26-OE5, TaPKS26-OE8, TaPKS26-OE9) as the test group and Fielder wheat as the control group, plants in both groups were inoculated with leaf rust fungi THT and PHT to detect the resistance of TaPKS26 overexpression lines to THT and PHT.

[0040] Following the inoculation methods in the national standard GB / T 45211.2-2025 "Technical Specification for Evaluation of Wheat Disease and Pest Resistance Part 2: Leaf Rust", T3 generation TaPKS26 overexpressing lines (TaPKS26-OE5, TaPKS26-OE8, TaPKS26-OE9) and Fielder wheat were inoculated with leaf rust fungi THT and PHT after the second leaf unfolded. Leaf phenotypes of each group were observed 12 days post-inoculation. Figure 3 As shown in A, after inoculation with leaf rust fungi THT and PHT, the number of spore masses on the leaves of the TapKS26 overexpressing line was significantly reduced compared with the Fielder line, indicating that the TapKS26 overexpressing line was more resistant to leaf rust.

[0041] Example 4

[0042] This embodiment detects the expression levels of disease resistance-related marker genes in TaPKS26 overexpression lines.

[0043] T3 generation TaPKS26 overexpression lines (TaPKS26-OE5, TaPKS26-OE8, TaPKS26-OE9) and Fielder plants were inoculated with leaf rust fungi THT and PHT after the second leaf unfolded. Samples were collected at 24 h and 48 h after inoculation to detect the relative expression levels of TaPR1, TaPR2 and TaPR5 genes in the samples.

[0044] Leaves from the inoculation site of wheat plants were cut, wrapped in aluminum foil, and flash-frozen in liquid nitrogen, then stored at -80℃ 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. The relative expression levels of TaPR1, TaPR2, and TaPR5 genes were then detected using qRT-PCR. Specific qRT-PCR primers were designed based on the sequences of the disease resistance-related marker genes TaPR1, TaPR2, TaPR5, and the elongation factor gene TaEF. The primer pairs for TaPR1 were TaPR1-F and TaPR1-R, for TaPR2 were TaPR2-F and TaPR2-R, and for TaPR5 were TaPR5-F and TaPR5-R. TaEF was used as an internal control, with the primer pairs being TaEF-F and TaEF-R. The sequence information of the above primers is shown in Table 1.

[0045] 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 cDNA from different sampling sites for each strain as templates, following the manufacturer's instructions. Each gene was performed in triplicate. The Ct values, mean, and standard deviations of each replicate were generated by the quantitative PCR instrument after manually adjusting the baseline. Two [units / items] were used. -ΔΔCt The relative expression levels of disease resistance-related marker genes TaPR1, TaPR2, and TaPR5 were determined by analyzing experimental data using a relative quantitative algorithm.

[0046] The results of qRT-PCR are as follows Figure 3 As shown in B, C, and D, Figure 3 Figure B shows the relative expression levels of TaPR1 in the TapKS26 overexpressing line and Fielder plants. Figure 3 The graph in C represents the relative expression levels of TaPR2 in the TapKS26 overexpressing line and Fielder plants. Figure 3 Figure D shows the relative expression levels of TaPR5 in TaPKS26 overexpressing lines and Fielder plants. The results showed that, compared to the control Fielder, the T3 generation TaPKS26 overexpressing lines exhibited significantly upregulated relative expression levels of disease resistance-related marker genes TaPR1, TaPR2, and TaPR5 at 24 and 48 hours after inoculation with leaf rust fungus THT and PHT, indicating that TaPKS26 overexpressing lines enhance plant resistance to leaf rust fungus.

[0047] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. The application of wheat disease resistance factor TaPKS26 in the control of leaf rust, characterized in that, Overexpression of the wheat disease resistance factor TaPKS26 in wheat enhances wheat resistance to leaf rust. The amino acid sequence of the wheat disease resistance factor TaPKS26 is shown in SEQ ID NO:2, and the CDS sequence of the gene of the wheat disease resistance factor TaPKS26 is shown in SEQ ID NO:

1.

2. A method for enhancing wheat resistance to leaf rust, characterized in that, Includes a gene that overexpresses the wheat disease resistance factor TaPKS26 in wheat plants, the CDS sequence of which is shown in SEQ ID NO:

1.

3. The method for enhancing wheat resistance to leaf rust according to claim 2, characterized in that, A recombinant overexpression vector was constructed and transformed into wheat. The recombinant overexpression vector contained the gene for the wheat disease resistance factor TaPKS26.

4. A method for breeding wheat varieties resistant to leaf rust, characterized in that, The gene containing the wheat disease resistance factor TaPKS26 overexpressed in wheat was obtained. TaPKS 26 overexpressing plants, the CDS sequence of the wheat disease resistance factor TaPKS26 gene is as shown in SEQ ID NO:

1.

5. The cultivation method according to claim 4, characterized in that, Gene infection with wheat leaf rust pathogen TaPKS When 26 plants are overexpressed, disease resistance marker genes are present. TaPR1 , TaPR2 and TaPR5 Expression was upregulated in all overexpression plants.

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