Application of the wheat stripe rust effector protein gene HASP 38 in improving wheat stripe rust resistance

Abstract

The application belongs to the field of bio-agriculture, and particularly relates to a wheat stripe rust effector protein gene HASP 38 and application thereof in improving wheat resistance to stripe rust. The amino acid sequence of the wheat stripe rust effector protein HASP 38 provided by the application is shown as SEQ ID NO: 1, the nucleotide sequence of the wheat stripe rust effector protein gene is shown as SEQ ID NO: 2, and an RNA interference vector of the gene is provided. HASP 38 The application transforms the RNA interference vector of the gene into Fielder wild-type material to obtain a gene-silenced wheat which exhibits resistance to stripe rust CYR32. HASP 38 The application determines that the wheat stripe rust effector protein gene HASP 38 plays a negative regulation role in wheat resistance to stripe rust, and uses the negative regulation factor to create a wheat material resistant to stripe rust, thereby providing a new gene and germplasm resource for wheat breeding against stripe rust.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, specifically relating to the wheat stripe rust effector protein gene. HASP 38 Application in improving wheat resistance to stripe rust. Background Technology

[0002] Puccinia striata wheat specialist type ( Puccinia striiformis f. sp. tritici , PST ) is a live-vegetative obligate parasitic fungus that infects wheat ( Triticum aestivum Wheat stripe rust, caused by *L.*, is known as "wheat cancer" and is one of the major diseases affecting wheat production. It can occur from wheat emergence to maturity and is widely distributed globally. In typical epidemic years, it can lead to a 10%–20% yield reduction, and in severe cases, over 60%. Wheat stripe rust often breaks out on a large scale, primarily because the urediniospores of *L.* can be spread over long distances via air currents, making it a typical airborne disease. Planting resistant varieties is the most economical and effective measure for controlling wheat stripe rust. Because *L.* can continuously produce new virulent races through mutation, vertical resistance in varieties is easily "lost," and since 1950, there have been several changes in dominant races, making the control of wheat stripe rust even more difficult. At the same time, traditional rust-resistant breeding faces the challenges of limited resistant germplasm resources and insufficient gene resource mining. Therefore, researching the virulence mechanism of *L.* and controlling wheat stripe rust is urgently needed.

[0003] Pathogens can interfere with host immunity and promote host infection by secreting small molecules—effect proteins. This induces effector-triggered susceptibility (ETS) in the host. Effector proteins can regulate the host's immune response in various ways to promote pathogen infection and are important virulence factors for pathogens. For example, wheat stripe rust fungus can affect electron transport in the photosystem through effector proteins, thereby inhibiting host photosynthesis and ROS production, creating an environment favorable for its own survival. Effector proteins utilize host susceptibility genes to promote host infection, enhance susceptibility gene kinase activity and nuclear translocation, and reduce the expression of downstream defense-related genes in the cell nucleus. Effector proteins can also interfere with the response of defense genes by influencing host alternative splicing. In plants, the NLR (Neuro-Linger) acts as an intracellular immune receptor, recognizing these effector proteins. In the absence of pathogens, the NLR remains in a state of self-inhibition through various intermolecular or intramolecular interactions. Once a pathogen is recognized, these interactions are disrupted, and the plant rapidly activates an immune response, including the expression of defense genes and the release of reactive oxygen species (ROS). This is often accompanied by a reactive oxygen species (HR) response, activating a stronger and more robust effector-triggered immunity (ETI). Therefore, identifying new pathogen effector proteins and elucidating their mechanisms of action is of great significance for the control of plant diseases and lays a theoretical foundation for the breeding of disease-resistant varieties. Summary of the Invention

[0004] The purpose of this invention is to study the effector protein gene of wheat stripe rust fungus. HASP 38 Exploring the role and mechanism of wheat stripe rust in wheat infection, and investigating the genes of wheat stripe rust effector proteins. HASP 38 The application of wheat stripe rust effector protein HASP 38 in the creation of plant disease-resistant varieties solves the technical problems of reproductive isolation and incompatibility of distant hybridization in traditional disease-resistant breeding, which makes it difficult to achieve targeted improvement of target traits within a short breeding cycle.

[0005] To ensure a complete and unambiguous understanding of the technical solution of this invention, it should be further noted that the wheat stripe rust effector protein HASP 38 encoding gene described in this invention is indicated by italicized font. HASP 38 The wheat stripe rust effector protein HASP 38 is indicated by the non-italicized font "HASP 38". 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.

[0006] On the one hand, this invention provides a wheat stripe rust effector protein gene. HASP 38 The wheat stripe rust effector protein gene HASP 38 The coding region nucleotide sequence is shown in SEQ ID NO:2.

[0007] Secondly, the present invention also provides a wheat stripe rust effector protein HASP 38, wherein the wheat stripe rust effector protein HASP 38 is derived from the wheat stripe rust effector protein gene. HASP 38 The amino acid sequence of the wheat stripe rust effector protein HASP 38 is shown in SEQ ID NO:1.

[0008] Thirdly, the present invention also provides an RNA interference vector, which is inserted into the wheat stripe rust effector protein gene. HASP 38 The gene silencing fragment, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0009] Fourthly, the present invention also provides a wheat stripe rust effector protein gene. HASP 38 The application of wheat stripe rust effector protein HASP 38 or the aforementioned RNA interference vector in improving wheat resistance to stripe rust.

[0010] Furthermore, in the application, the improvement is to cultivate wheat varieties resistant to stripe rust.

[0011] Furthermore, in the aforementioned application, the expression of the RNA interference vector through Agrobacterium-mediated genetic transformation plays a negative regulatory role in wheat's resistance to stripe rust infection.

[0012] Furthermore, in this application, the wheat stripe rust effector protein gene is silenced. HASP 38 Enhance wheat's resistance to stripe rust pathogen.

[0013] Fifthly, the present invention also provides a method for breeding wheat varieties resistant to stripe rust, wherein the method involves transforming the aforementioned RNA interference vector within the plant to obtain... HASP 38 Gene-silencing plant varieties.

[0014] Furthermore, the method for breeding wheat varieties resistant to stripe rust includes transforming the immature embryos of the plant using Agrobacterium-mediated genetic transformation to obtain... HASP 38 Gene-silencing plant varieties.

[0015] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0016] (1) Compared with traditional disease-resistant breeding techniques, plant disease-resistant genetic engineering technology can overcome reproductive isolation and incompatibility between species, achieving targeted improvement of target traits in a shorter time and providing crops with more comprehensive, continuous, and broad-spectrum protection. This invention, through gene function research, discovered the wheat stripe rust effector protein gene. HASP 38 It plays a negative regulatory role in the immune defense response of wheat against stripe rust infection, namely, silencing wheat stripe rust effector protein genes. HASP 38 It can improve wheat's resistance to stripe rust. It reduces the effector protein of wheat stripe rust fungus. HASP 38 The expression can endow plants with a certain degree of disease resistance.

[0017] (2) This invention provides a method for breeding wheat varieties resistant to stripe rust. This method uses RNA interference technology to transform the aforementioned RNA interference vector into wheat plants, thereby enhancing the wheat's resistance to stripe rust pathogens. Verification has shown that the wheat varieties obtained using this method... HASP 38 The gene-silenced wheat strain exhibits resistance to the main prevalent races of stripe rust. This invention provides a new technical approach for breeding wheat varieties resistant to stripe rust from a molecular biology perspective, and effectively solves the technical problems of this invention. Attached Figure Description

[0018] Picture 1 It is the wheat stripe rust effector protein gene. HASP 38 Schematic diagram of expression profile analysis of stripe rust race CYR31 (affinity).

[0019] Picture 2 It is the wheat stripe rust effector protein gene. HASP 38 A schematic diagram of an RNA interference vector. LB and RB are homologous arms; Ubi is the promoter. HASP 38 The gene encoding HASP 38 is ; Act1 is the termination element; and Bar is the herbicide selection marker gene.

[0020] Picture 3 This is the PC336 vector map, used to construct effector protein genes. HASP 38 RNA interference vector.

[0021] Picture 4 It is the T0 generation. HASP 38 PCR identification results of gene-silenced plants. HASP 38 is... HASP 38 Lines of gene-silenced plants; M for DNA Marker; WT for wild type; Plasmid for plasmid-positive control group; H2O for water control group.

[0022] Picture 5 It's the T1 generation. HASP 38 Phenotypic results of gene-silenced plants inoculated with stripe rust fungus CYR32. HASP38 RNAi#L1 and HASP38 RNAi#L3 is the T1 generation. HASP 38 Gene-silenced plants, CYR32 indicates inoculation with stripe rust pathogen CYR32, Fielder is a wild-type wheat variety. 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 example relates to the wheat stripe rust effector protein gene. HASP 38 Functional analysis of the interaction between wheat and stripe rust.

[0028] qRT-PCR was used to determine the effector proteins. HASP 38 Expression of the elongation factor gene in the wheat-striped rust interaction. TaEF1-α As an internal reference, the wheat stripe rust effector protein gene was utilized. HASP 38 qRT-PCR was performed using specific primers to identify the wheat stripe rust effector protein gene. HASP 38 Expression levels of wheat stripe rust effector proteins at different time points after infection with wheat. qRT-PCR was used to detect the expression levels of wheat stripe rust effector proteins at different time points (6 h, 12 h, 24 h, 48 h, 72 h, and 180 h after infection) when wild-type wheat was inoculated with stripe rust CYR31 (a compatible race) in a water source. HASP 38 The induced expression status. In qRT-PCR HASP 38 Quantitative primers use primer pairs HASP 38 -qRT-F and HASP 38 -qRT-R, internal parameter TaEF1-α Primers use primer pairs TaEF1α- F sum TaEF1α -R, the primer information is shown in the table.

[0029] Table 1 Primer sequence information

[0030]

[0031] qRT-PCR test results are as follows Picture 1 As shown, the wheat stripe rust effector protein gene is displayed. HASP 38 Expression levels reached their peak after 6 hours of infection in the affinity system (inoculated with CYR31), indicating that the wheat stripe rust effector protein gene... HASP 38It is induced by wheat stripe rust and participates in the wheat-stripe rust interaction.

[0032] Example 2

[0033] This embodiment provides a wheat stripe rust effector protein gene. HASP 38 Application in the improvement of wheat rust-resistant varieties.

[0034] In this embodiment, the plant is preferably a monocotyledonous cereal crop that can be successfully infected and colonized by wheat stripe rust pathogen, with wheat being particularly preferred.

[0035] The wheat stripe rust effector protein gene in this embodiment HASP 38 The nucleotide sequence is shown in SEQ ID NO:2.

[0036] The amino acid sequence of the wheat stripe rust effector protein HASP 38 in this embodiment is shown in SEQ ID NO:1.

[0037] The wheat stripe rust effector protein gene provided in this embodiment HASP 38 Applications in improving wheat rust-resistant varieties include the creation of varieties using RNA interference technology. HASP 38 Gene-silenced plants.

[0038] Bioinformatics analysis of wheat stripe rust effector protein genes HASP 38 Based on the nucleotide characteristics, the wheat stripe rust effector protein gene was selected. HASP 38 The gene silencing fragment, the selected gene HASP 38 The nucleotide sequence of the gene silencing fragment is shown in SEQ ID NO:3, and it is used to construct the wheat stripe rust effector protein gene. HASP 38 RNA interference vector. Primers were used. HASP 3 8-DONR221-F and HASP 3 8-DONR221-R was amplified by PCR, and the PCR product was recovered by agarose gel electrophoresis and gel excision, yielding samples with attB1 and attB2 sites at both ends. HASP 38 Gene silencing fragments. Through the BP response, [the following is observed / implanted / discontinued / etc.] HASP 38 The gene-silenced fragment was ligated into the intermediate vector pDONR221 and transformed into *E. coli* TOP10 strain. The transformed strain was plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies were picked and tested for positivity using primers M13-F and M13-R. Positive colonies were marked, cultured, and then subjected to plasmid extraction and sequencing. HASP 38 The pDONR221 vector containing the gene silencing fragment was digested with NruI restriction enzyme, then separated by agarose gel electrophoresis, and the linearized vector was recovered. The obtained linearized vector and the final vector PC336 were then subjected to an LR reaction. HASP 38 The gene-silenced fragment was ligated into the final vector PC336 and transformed into *E. coli* TOP10 strain. The culture was plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies were picked and tested for positivity using primers PC336-F and PC336-R. Positive colonies were marked, and plasmids were extracted and sequenced. Sequencing results were compared with the silenced fragment; vectors whose sequencing results matched the silenced fragment were considered successfully constructed. HASP 38 The RNA interference vector. Primer information used in this implementation is shown in Table 1.

[0039] HASP 38 A schematic diagram of the RNA interference vector is shown below. Picture 2 As shown, Picture 2 middle HASP 38 for HASP 38 Silent segments of genes ， Its nucleotide sequence is shown in SEQ ID NO:3, and the RNA interference vector PC336 vector map is shown in [image missing]. Picture 3 As shown.

[0040] Prepared HASP 38 The RNA interference vector was introduced into Fielder wheat cells using Agrobacterium-mediated transformation to obtain... HASP 38 T0 generation of gene-silenced plants. A total of 12 plants were obtained. HASP 38 Gene-silenced plants.

[0041] The wheat stripe rust effector protein gene provided in this embodiment HASP 38 Its application in improving wheat rust-resistant varieties also includes wheat stripe rust effector protein genes. HASP 38 The verification methods for its application in breeding and improving wheat rust-resistant varieties specifically include:

[0042] S101, obtain HASP 38 Gene-silenced plants, for the obtained HASP 38 Molecular detection was performed on gene-silenced plants;

[0043] S102, compared to the T1 generation HASP 38 Gene-silenced plants were inoculated with the prevalent stripe rust race CYR32, and the resistance of gene-silenced plants to the prevalent stripe rust race was identified.

[0044] Detection using PCR technology HASP 38 Gene-silencing positive plants were analyzed by PCR products using 1% agarose gel electrophoresis. Results are as follows: Picture 4 As shown, Picture 4 M stands for DNA Marker; HASP38 is... HASP 38Lines of gene-silencing positive plants; WT was the wild type (an empty lane was set between HASP38 and WT for easier observation of electrophoresis results); Plasmid was the plasmid-positive control group; H2O was the water control group. Results showed that the T0 generation yielded a total of [number missing] lines. HASP 38 Twelve gene-silencing positive plant lines were identified.

[0045] This embodiment provides a wheat stripe rust effector protein gene. HASP 38 Application in improving wheat varieties resistant to rust. The T1 generation was selected. HASP 38 Gene-silenced Line 1 strains and HASP 38 In wheat plants with the gene-silenced Line3 line, when they reached the "two-leaf-one-heart" stage, the two leaves were inoculated with stripe rust fungus CYR32. Wild wheat (Fielder) served as the control group and was also inoculated with stripe rust fungus CYR32. The growth of spore masses on wheat leaves was observed 14 days after inoculation, and the results are as follows: Picture 5 As shown, HASP 38 Gene-silenced Line 1 strains ( HASP38 RNAi#L1) and HASP 38 Gene-silenced Line3 strains ( HASP38 RNAi#L3 produced significantly fewer sporulations than wild-type plants (Fielder). The results indicated... HASP 38 Gene-silenced plants exhibit enhanced resistance to stripe rust.

[0046] In summary, the embodiments of the present invention provide a gene for the effector protein of wheat stripe rust fungus. HASP 38 Application in improving wheat rust-resistant varieties: utilizing gene recombination technology to construct genes. HASP 38 RNA interference vectors were used to induce gene transfer in wheat through Agrobacterium-mediated transformation. HASP 38 RNA interference vectors were delivered to recipient wheat Fielder cells, and gene-silenced plants were obtained and identified using PCR detection technology. HASP 38 Gene-silencing positive plants were selected. HASP 38 Two lines, L1 and L3, of the T1 generation of gene-silenced plants were inoculated with the prevalent race CYR32. After 14 days, observation revealed the presence of the wheat stripe rust effector protein gene. HASP 38 Compared with Fielder, the gene-silenced plants produced significantly less sporulation and showed enhanced resistance to wheat stripe rust.

[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. Application of RNA interference vector in wheat stripe rust improvement, wherein the RNA interference vector is inserted into the wheat stripe rust effector protein gene. HASP 38 The gene silencing fragment, the nucleotide sequence of which is shown in SEQ ID NO:

3.

2. The application according to claim 1, characterized in that, The improvement is aimed at developing wheat varieties resistant to stripe rust.

3. The application according to claim 2, characterized in that, The RNA interference vector, expressed through Agrobacterium-mediated genetic transformation, plays a negative regulatory role in wheat's resistance to stripe rust infection.

4. Silencing wheat stripe rust effector protein genes HASP 38 Its application in the improvement of wheat stripe rust is characterized by... The wheat stripe rust effector protein gene HASP 38 The coding region nucleotide sequence is shown in SEQ ID NO:2, silencing the wheat stripe rust effector protein gene. HASP 38 Enhance wheat's resistance to stripe rust pathogens.

5. A method for breeding wheat varieties resistant to stripe rust, characterized in that, Transformation of RNA interference vectors within plants ， get HASP 38 In gene-silencing plant varieties, the RNA interference vector is inserted into the wheat stripe rust effector protein gene. HASP 38 The gene silencing fragment, the nucleotide sequence of which is shown in SEQ ID NO:

3.

6. The method for breeding wheat varieties resistant to stripe rust according to claim 5, characterized in that, This includes transforming the immature embryos of the plant using Agrobacterium-mediated genetic transformation to obtain... HASP 38 Gene-silencing plant varieties.

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