Application of gene TaToc34 and encoded protein thereof in regulation and control of wheat stripe rust resistance

Abstract

The invention belongs to the technical field of biological agriculture, and discloses application of a gene TaToc34 and an encoding protein thereof in regulation and control of wheat stripe rust resistance. The nucleotide sequence of the gene TaToc34 is as shown in SEQ ID NO: 1, the amino acid sequence of the protein TaToc34 is as shown in SEQ ID NO: 2, tests verify that the gene TaToc34 is subjected to induced expression of stripe rust, an agrobacterium-mediated method is adopted to construct a gene silencing plant of the gene TaToc34, the gene TaToc34 plays a positive regulation role in immune response of wheat to stripe rust, the silencing gene TaToc34 reduces expression of wheat defensive genes, and the gene TaToc34 has a positive regulation role in immune response of wheat to stripe rust. The wheat stripe rust resistance is reduced, and a new gene resource is provided for improvement of the wheat stripe rust.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, specifically relating to the application of the gene TaToc34 and its encoded protein in regulating wheat stripe rust resistance. Background Technology

[0002] During wheat production, various pathogens affect wheat yield. Among them, wheat stripe rust has a particularly significant impact. Its pathogen, Puccinia striiformis f.sp. tritici (Pst), is widely distributed in major wheat-producing areas and is the main cause of a sharp decline in wheat yield. In recent years, new, highly virulent and invasive stripe rust races have emerged and spread rapidly and frequently, triggering several large-scale epidemics of wheat stripe rust.

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

[0004] Chloroplasts, as the core organelles for photosynthesis in plants, play a crucial role in maintaining photosynthetic efficiency and coping with various adverse environmental conditions through their protein transport mechanisms. The Toc complex is a key component in chloroplast protein transport, containing at least four proteins involved in protein transport: two receptor proteins with GTPase activity, Toc159 and Toc34, and transport channel proteins, Toc75 and Toc64. Among these, Toc34 plays a critical role in chloroplast protein transport, primarily responsible for recognizing and transporting nuclear-encoded precursor proteins into the chloroplast.

[0005] Although the basic protein transport function of Toc34 has been studied to some extent, its role and mechanism in plant response to adversity, biotic stress, and abiotic stress are rarely reported. In-depth research into the function of the wheat chloroplast transport enzyme gene TaToc34 in plant immunity is expected to provide new ideas and technical means for the control of wheat stripe rust. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention employs genetic engineering techniques and genetic research tools to study the function of the wheat chloroplast transporter gene TaToc3 and its protein under biological stress, providing new genetic resources for the green control of wheat stripe rust.

[0007] To ensure a complete and unambiguous understanding of the technical solution of this invention, it should be noted that the wheat chloroplast transporter gene TaToc3 is represented by italicized text "gene TaToc3", while the wheat chloroplast transporter protein TaToc3 is represented by non-italicized text "protein TaToc3". 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.

[0008] On the one hand, the present invention provides the application of gene TaToc34 in regulating wheat stripe rust resistance, wherein gene TaToc34 positively regulates wheat stripe rust resistance, and the nucleotide sequence of gene TaToc34 is shown in SEQ ID NO:1.

[0009] Secondly, the present invention also provides the application of protein TaToc34 in regulating wheat stripe rust resistance, wherein protein TaToc34 is encoded by gene TaToc34, the amino acid sequence of protein TaToc34 is shown in SEQ ID NO:2, and protein TaToc34 positively regulates wheat stripe rust resistance.

[0010] Thirdly, the present invention provides a method for regulating wheat resistance to stripe rust by silencing the TaToc34 gene in wheat to reduce wheat resistance to stripe rust.

[0011] Fourthly, the present invention provides a method for regulating wheat resistance to stripe rust by reducing the expression level or activity of protein TaToc34 in wheat, thereby reducing wheat resistance to stripe rust.

[0012] Fifthly, the present invention also provides a method for breeding wheat varieties susceptible to stripe rust, comprising silencing the TaToc34 gene in wheat to obtain TaToc34-silenced plants.

[0013] Furthermore, in the method, a recombinant vector containing a TaToc34 gene silencing fragment is constructed, and the recombinant vector containing the TaToc34 gene silencing fragment is transformed into wheat cells using Agrobacterium-mediated transformation to obtain TaToc34 gene silencing plants.

[0014] Furthermore, in the method, the nucleotide sequence of the TaToc34 gene silencing fragment is shown in SEQ ID NO:3.

[0015] Furthermore, in the method, when the wheat stripe rust invasion gene TaToc34 is silenced in plants, the expression of wheat defense genes is downregulated.

[0016] Furthermore, in the method, the wheat defense genes include TaPR1, TaPR2, and TaWRKY53.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0018] This invention discovers that the wheat chloroplast transporter gene TaToc3 participates in plant immune regulation. It was found that silencing the wheat chloroplast transporter gene TaToc3 downregulates the expression of wheat defense genes TaPR1, TaPR2 and TaWRKY53, weakening wheat's resistance to stripe rust and providing new gene resources for the green control of wheat stripe rust. Attached Figure Description

[0019] Figure 1 A schematic diagram showing the expression profiles of the TaToc34 gene at different time points after inoculating Shuiyuan 11 wheat with both the compatible race (CYR32) and the incompatible race (CYR23).

[0020] Figure 2 This image shows the PCR identification results of plants with the TaToc34-RNAi gene. Lane M represents the DNA 2000 Marker; lane TaToc34-RNAi represents plants with the TaToc34-RNAi gene, of which there are 6 plants: L6, L5, L4, L3, L2, and L1; lane WT represents the negative control of Fielder wild-type plants; and lane ddH2O represents the blank control of ddH2O.

[0021] Figure 3 Figure A shows the results of disease resistance identification in TaToc34-RNAi plants. Figure B shows the phenotype of wheat plants after inoculation with stripe rust fungus CYR23; Figure C shows the statistical results of the relative biomass of stripe rust fungus in wheat plants inoculated with stripe rust fungus CYR23; Figure C shows the statistical results of the relative expression level of gene TaToc34 in TaToc34-RNAi plants.

[0022] Figure 4 Figure 1 shows the relative expression levels of wheat defense genes after TaToc34-RNAi plants were inoculated with wheat stripe rust fungus CYR23. Figure 2 shows the relative expression levels of wheat defense gene TaPR1; Figure 3 shows the relative expression levels of wheat defense gene TaPR2; Figure 4 shows the relative expression levels of wheat defense gene TaWRKY53. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] 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.

[0026] Example 1

[0027] This embodiment provides the amplification and sequencing of the wheat chloroplast transporter gene TaToc34.

[0028] Full-length primers for the wheat chloroplast transporter gene TaToc34 were designed, including a forward primer TaToc34-F and a reverse primer TaToc34-R. Primer sequence information is shown in Table 1. Using cDNA from wheat plants of the Shuiyuan 11 origin as a template, the full-length wheat chloroplast transporter gene TaToc34 was amplified, constructed into the pMD-19T vector, and sequenced. The nucleotide sequence of the wheat chloroplast transporter gene TaToc34 is shown in SEQ ID NO:1, and the amino acid sequence of the wheat chloroplast transporter protein TaToc34 is shown in SEQ ID NO:2.

[0029] Table 1 Primer sequence list

[0030]

[0031]

[0032] Example 2

[0033] This embodiment aims to detect the relative expression level of the wheat chloroplast transporter gene TaToc34 during the interaction between wheat and stripe rust fungus.

[0034] Wheat seedlings (Wheat 11) at the two-leaf stage were inoculated with the compatible race CYR32 and the incompatible race CYR23. Data were collected at 0, 6, 12, 24, 48, 72, 96, and 120 hpi after inoculation. Total RNA was extracted from the collected wheat leaves using a plant RNA extraction kit from Beijing Huayueyang Biotechnology Co., Ltd., and the extracted RNA was reverse transcribed into cDNA using a Thermo reverse transcription kit.

[0035] Specific quantitative PCR primers were designed based on the wheat chloroplast transporter TaToc34 gene sequence. The TaToc34 gene quantitative PCR primers are TaToc34-qRT-F and TaToc34-qRT-R. The wheat elongation factor gene TaEF was used as an internal reference gene, and the TaEF gene primers are TaEF-qRT-F and TaEF-qRT-R. The primers mentioned above and the primer information used below are shown in Table 1.

[0036] Following the operating instructions of the UltraSYBR One Step RT-qPCR Kit from Kangwei Century Biotechnology Co., Ltd., RT-qPCR was performed using cDNA from different time points as templates.

[0037] RT-qPCR amplification program: The initial steps are 95℃ for 5 min, 94℃ for 5 s, 60℃ for 30 s, repeated 40 times, followed by a final temperature drop of 0.5℃ for 0.5 s. -1 The temperature was increased from 60°C to 95°C, and the instrument automatically collected the cycle threshold (Ct) value. Each reaction was repeated 3 times, using 2 -ΔΔCt The relative expression levels of genes were determined. Each biological sample was tested three times, and the t-test was used to assess statistical significance.

[0038] The test results are as follows Figure 1 As shown, the wheat chloroplast transporter gene TaToc34 was highly induced by stripe rust fungus at 48 hpi in the affinity combination, and highly induced by stripe rust fungus at 6 hpi in the incompatible combination, indicating that the wheat chloroplast transporter gene TaToc34 is induced by stripe rust fungus.

[0039] Example 3

[0040] This example demonstrates the construction of a TaToc34-RNAi gene plant.

[0041] Primers TaToc34-PC336-F and TaToc34-PC336-R were designed based on a specific fragment of the wheat chloroplast transporter gene TaToc34.

[0042] Using the full-length TaToc34 gene amplified in Example 1 as a template, PCR amplification was performed using primers TaToc34-PC336-F and TaToc34-PC336-R, followed by gel extraction to obtain the target fragment. The target fragment was ligated into the pDONR221 vector via a backpropagation (BP) reaction at 25°C for 8 hours. After the reaction, the fragment was transformed into DH5α competent cells, and the plasmid was extracted. The obtained TaToc34-specific fragment-pDONR221 recombinant vector was digested with NruI at 37°C for 1 hour. The digestion product was then gel-extracted and ligated into the PC336 vector via a backpropagation (LR) reaction at 25°C for 8 hours. After the reaction, the fragment was transformed into DH5α competent cells, and the plasmid was extracted and sequenced. The recombinant vector with correct sequencing results was transformed into EHA105 Agrobacterium competent cells, and subsequently, Agrobacterium-mediated transformation of wheat embryos was used to obtain TaToc34-RNAi wheat.

[0043] The obtained TaToc34-RNAi wheat plants were planted in pots containing nutrient soil. During the four-leaf stage, leaves from all TaToc34-RNAi wheat plants and wild-type wheat were collected. Genomic DNA was extracted from the leaves using the CTAB method, and then PCR detection was performed using the extracted genomic DNA as a template. Primers PC366-F and PC366-R from the PC336 vector were used for detection. Wild-type wheat was used as a negative control, and ddH2O was used as a blank control. The PCR results were detected by electrophoresis. The electrophoresis results are shown below. Figure 2 As shown.

[0044] Depend on Figure 2 It can be seen that no corresponding band was detected in wild type and ddH2O, but a corresponding band was detected in wheat TaToc34-RNAi transgenic plants at around 250bp, indicating that the TaToc34-PC336 recombinant vector was transformed into wheat, and a total of 6 T0 generation positive TaToc34-RNAi plants were obtained: L1, L2, L3, L4, L5 and L6.

[0045] Example 4

[0046] This example demonstrates the detection of the relative expression of the TaToc34 gene in TaToc34-RNAi plants.

[0047] T1 generation TaToc34-RNAi#L3 and TaToc34-RNAi#L5 wheat plants (referred to as L3 and L5) and wild-type wheat (WT) were selected and grown in a greenhouse. When they grew to the stage of two leaves and one bud, they were inoculated with wheat stripe rust fungus CYR23. Phenotypic characteristics were observed 14 days after inoculation. The phenotypes are as follows: Figure 3As shown in Figure A, after inoculation with CYR23, wild-type wheat leaves produced a large number of necrotic spots but no spores. The leaves of TaToc34-RNAi (L3 and L5) plants not only produced necrotic spots but also produced a small number of spores, indicating that the TaToc34-RNAi plants had weakened resistance to stripe rust.

[0048] DNA was extracted from the phenotypic leaves collected and observed above using the CTAB method described in Example 3. The relative biomass of stripe rust fungi was detected by qRT-PCR, and the results are as follows: Figure 3 As shown in B, compared with the control group (WT), the relative biomass of stripe rust fungus in the leaves of TaToc34-RNAi#L3 and TaToc34-RNAi#L5 plants was significantly increased, indicating that the TaToc34-RNAi gene plants had weakened resistance to stripe rust fungus.

[0049] Before inoculation with CYR23 stripe rust, two-leaf leaves of TaToc34-RNAi#L3 and TaToc34-RNAi#L5 wheat and control wild-type wheat (WT) were collected. RNA was extracted according to the method in Example 2, and the relative expression level of the TaToc34 gene was detected. The results are as follows: Figure 3 As shown in C, compared with the control group, the expression level of gene TaToc34 was significantly reduced in TaToc34-RNAi#L3 and TaToc34-RNAi#L5 plants.

[0050] Example 5

[0051] This example is used to detect the relative expression level of wheat defense genes in TaToc34-RNAi plants.

[0052] Following the method described in Example 4, TaToc34-RNAi plants were inoculated with CYR23. Samples were collected at 24 hpi, and RNA was extracted according to the method described in Example 2. The RNA was then reverse transcribed into cDNA, and RT-qPCR was performed to detect the relative expression levels of wheat defense genes. The wheat defense genes include TaPR1, TaPR2, and TaWRKY53. The specific primers for TaPR1 are TaPR1-qRT-F and TaPR1-qRT-R; the specific primers for TaPR2 are TaPR2-qRT-F and TaPR2-qRT-R; and the specific primers for TaWRKY53 are TaWRKY53-qRT-F and TaWRKY53-qRT-R.

[0053] Test results as follows Figure 4As shown, compared with the control wild-type wheat (WT), the relative expression levels of wheat defense genes TaPR1, TaPR2 and TaWRKY53 were significantly reduced in TaToc34-RNAi#L3 and TaToc34-RNAi#L5 plants, indicating that the silencing gene TaToc34 weakens wheat's resistance to stripe rust.

[0054] 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 the TaToc34 gene in regulating wheat stripe rust resistance, characterized in that, The gene TaToc34 positively regulates wheat stripe rust resistance, and the nucleotide sequence of the gene TaToc34 is shown in SEQ ID NO:

1.

2. The application of protein TaToc34 in regulating wheat stripe rust resistance, characterized in that, The protein TaToc34 is encoded by the gene TaToc34, and the amino acid sequence of the protein TaToc34 is shown in SEQ ID NO:

2. The protein TaToc34 positively regulates wheat stripe rust resistance.

3. A method for regulating wheat resistance to stripe rust, characterized in that, Silencing the TaToc34 gene in wheat reduces its resistance to stripe rust.

4. A method for regulating wheat resistance to stripe rust, characterized in that, Reducing the expression level or activity of the protein TaToc34 in wheat can decrease its resistance to stripe rust.

5. A method for breeding wheat varieties susceptible to stripe rust, characterized in that, This includes silencing the TaToc34 gene in wheat to obtain TaToc34-silenced plants.

6. The method according to claim 5, characterized in that, By constructing a recombinant vector containing a TaToc34 gene silencing fragment, the recombinant vector containing the TaToc34 gene silencing fragment was transformed into wheat cells using Agrobacterium-mediated transformation to obtain TaToc34 gene-silencing plants.

7. The method according to claim 6, characterized in that, The nucleotide sequence of the TaToc34 gene silencing fragment is shown in SEQ ID NO:

3.

8. The method according to claim 6, characterized in that, When the wheat stripe rust invasion gene TaToc34 is silenced in wheat plants, the expression of wheat defense genes is downregulated.

9. The method according to claim 8, characterized in that, The wheat defense genes include TaPR1, TaPR2, and TaWRKY53.

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