Application of effector protein HASP 97 in improving wheat disease resistance

Abstract

The present invention belongs to the field of genetic engineering technology and relates to the application of effector protein HASP 97 in improving wheat disease resistance. The present invention studies the function of effector protein HASP 97 by genetic means, performs transgenic in Fielder wild-type materials, and obtains HASP 97 Gene-silenced plants have been found HASP 97 Gene silenced plants showed enhanced resistance to stripe rust, and the effector protein HASP 97 was found to be induced to express during the infection of wheat by wheat stripe rust, confirming the gene HASP 97 The invention plays a negative regulatory role in wheat stripe rust resistance. The invention provides an effector protein for genetic engineering modification and lays the foundation for further breeding new materials or new varieties of wheat stripe rust resistance. It can also provide theoretical and technical support for the future use of the effector protein for plant genetic improvement.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to the application of effector protein HASP 97 in improving disease resistance of wheat. Background Art

[0002] Puccinia striiformis f. sp. tritici (Pst), an obligate parasite, infects wheat (Triticum aestivum L.), causing wheat stripe rust, a major disease in wheat production. Wheat stripe rust can occur from seedling emergence to maturity. It is a typical airborne disease, spread by the fungus on high-altitude air currents. Planting resistant varieties is the most cost-effective and effective prevention and control measure for wheat stripe rust. Pathogenic variation in wheat stripe rust constantly generates new strains, leading to a loss of vertical resistance in varieties. Repeated shifts in dominant races complicate wheat stripe rust prevention and control. Traditional rust-resistant breeding faces the challenges of limited germplasm resources and insufficient genetic resources.

[0003] Pathogens can secrete small proteins called effectors to interfere with host immunity, promote infection, establish affinity interactions with the host, and induce host susceptibility (ETS). Effectors can modulate host immune responses and promote pathogen infection in various ways, making them important virulence factors. Wheat stripe rust fungi use effectors to influence electron transport in the photosystem, thereby inhibiting host photosynthesis and ROS production, thereby promoting their own survival. Effector proteins exploit host susceptibility genes to promote infection, enhancing kinase activity and nuclear import, and reducing the expression of downstream defense-related genes in the nucleus. Effector proteins can also interfere with defense gene responses by affecting alternative splicing. NLRs in plants act as intracellular immune receptors to recognize these effector proteins. In the absence of pathogens, NLRs maintain an autoinhibited state through various inter- and intramolecular interactions. Upon pathogen recognition, these interactions are disrupted, and the plant rapidly activates an immune response, including the expression of defense genes and the production of reactive oxygen species (ROS), often accompanied by a HR response, resulting in a more robust and robust effector-triggered immunity (ETI). Therefore, identifying new pathogen effector proteins and elucidating their mechanisms of action are crucial for controlling plant diseases and lay the theoretical foundation for the development of disease-resistant varieties. Uncovering wheat disease-resistant genetic resources and accelerating the development and breeding of new stripe rust-resistant varieties is of far-reaching significance. Summary of the Invention

[0004] The purpose of the present invention is to explore its mechanism of action during the infection process of wheat stripe rust, improve the understanding of the pathogenesis of the pathogen, and solve the problems of reproductive isolation and distant hybridization incompatibility in traditional disease-resistant breeding, which makes it difficult to achieve targeted improvement of target traits within a short breeding cycle.

[0005] In order to fully understand the technical solution of the present invention without objection, it is necessary to supplement that the effector protein or wheat stripe rust effector protein of the present invention is represented by the non-italic font "HASP 97", and its encoding gene is represented by the italic font " HASP 97 Of course, those skilled in the art can clearly and completely understand the meaning and expression of the relevant genes and their encoded proteins based on the description of the present invention.

[0006] On the one hand, the present invention relates to the use of effector protein HASP 97 in improving wheat disease resistance, wherein the effector protein HASP 97 is HASP 97 Gene encoding, the amino acid sequence of the effector protein HASP 97 is shown in SEQ ID NO: 1;

[0007] Silencing, reducing or knocking out the HASP 97 Genes that enhance wheat's resistance to stripe rust.

[0008] SEQ ID NO: 1 is specifically shown below,

[0009] MIRLSMIALVCFALRCTLAAPSLDVSTAPIVQAIGSLANLPFQDLQPHKPFPVDSARTLNYLKHFDPLKHFPDEKIHTEVPGGKNVELQVPRQQNSRKEFKSNIMKPPQARM KMDRTRTPTQEEEIKINNILNNYPVGHAYQLTDTPEPVILAAPIFDFEYACGKDKIYGIDGALIKETGCFHWNGPCVEECLVEAYVDCLKGSSKSSKSKKSYDNSNYSRKII.

[0010] Furthermore, in the application provided by the present invention, the HASP 97 The nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0011] SEQ ID NO: 2 is specifically shown below,

[0012] .

[0013] Further, in the application provided by the present invention, silencing, reducing or knocking out the HASP 97 Genes that enhance wheat resistance to stripe rust.

[0014] Further, in the application provided by the present invention, silencing, reducing or knocking out the HASP 97 Gene that reduces sporulation of stripe rust.

[0015] Further, in the application provided by the present invention, the silencing HASP 97 The gene expression vector comprises the nucleotide sequence shown in SEQ ID NO: 2.

[0016] In another aspect, the present invention relates to a method for breeding a wheat rust-resistant variety, which silences, reduces or knocks out the HASP 97 gene, the HASP 97 The gene encodes the effector protein HASP 97, and the amino acid sequence of the effector protein HASP 97 is shown in SEQ ID NO: 1.

[0017] Furthermore, in the method for breeding wheat rust-resistant varieties provided by the present invention, the HASP 97 The nucleotide sequence of the gene is shown in SEQ ID NO: 2.

[0018] Furthermore, in the method for breeding wheat rust-resistant varieties provided by the present invention, the silencing HASP 97 The gene expression vector comprises the nucleotide sequence shown in SEQ ID NO: 2. The expression vector is introduced into wheat immature embryos to obtain a wheat variety resistant to stripe rust.

[0019] Those skilled in the art can easily mutate the nucleotide sequence encoding the effector protein HASP 97 of the present invention using known methods, such as directed evolution and point mutation. HASP 97 Nucleotides with 75% or greater identity to the nucleotide sequence of a gene are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the effector protein HASP 97 and have the same function.

[0020] As used herein, the term "identity" refers to sequence similarity to a naturally occurring nucleic acid sequence. "Identity" includes nucleotide sequences that are 75% or greater, 85% or greater, 90% or greater, or 95% or greater identical to a nucleotide sequence of the present invention encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess identity between related sequences.

[0021] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0022] The present invention silences, reduces or knocks out the HASP 97 The purpose of this gene is to reduce the activity and / or expression of the effector protein HASP97 in wheat. Those skilled in the art can achieve this goal by using methods well known in the art, such as VIGS, RNA interference, homologous recombination, and site-directed gene editing.

[0023] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0024] (1) Compared with traditional disease-resistant breeding technology, plant disease-resistant genetic engineering technology can break through the reproductive isolation and incompatibility of distant hybridization between species, achieve targeted improvement of target traits in a shorter period of time, and provide more comprehensive, sustainable and broad-spectrum protection for crops. Through genetic engineering research, the present invention found that the wheat stripe rust effector protein HASP 97 promotes the infection of stripe rust in the interaction between stripe rust and wheat. At the same time, the process of stripe rust infecting wheat also promotes the expression of effector protein HASP97, that is, silencing, reducing or knocking out the gene expression of wheat stripe rust effector protein HASP 97 can improve the infection ability of stripe rust on wheat. When the gene fragment of wheat stripe rust effector protein HASP 97 is silenced and expressed in plants, it can give plants a certain degree of disease resistance.

[0025] (2) The present invention provides a method for breeding wheat varieties resistant to stripe rust. This method uses genetic engineering technology to silence the gene in the wheat plant. HASP 97 Genes are being silenced to enhance wheat resistance to the stripe rust pathogen. Wheat grown using the gene-silencing method of the present invention to express the effector protein HASP 97 has been shown to exhibit enhanced resistance to the major prevalent races of the fungus. This invention provides a new technical approach for breeding wheat varieties resistant to stripe rust from a molecular biology perspective, effectively resolving the technical problems addressed by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Picture 1 Schematic diagram of expression profiling of effector protein HASP 97. CYR23 is a non-compatible race of stripe rust, while CYR31 is a compatible race of stripe rust.

[0028] Picture 2 The following are the PCR test results of wheat plants expressing the gene-silenced effector protein HASP 97. HASP 97 is the gene-silenced plant line; M is a DNA marker; WT is the wild type; Plasmid is the plasmid positive control group; and H2O is the water control group.

[0029] Picture 3 This is a diagram of the gene expression vector for the effector protein HASP 97. LB and RB are homology arms; Ubi is the promoter; HASP 97is the HASP 97 gene; Act1 is the termination element; and Bar is the herbicide selection marker gene.

[0030] Picture 4 This is the phenotypic result of the effector protein HASP 97 gene silenced plants inoculated with stripe rust fungus CYR32. HASP97 RNAi#L3 and HASP97 RNAi #L7 indicates the effector protein HASP97 gene silenced plant, CYR32 indicates the inoculation with the virulent stripe rust race CYR32, and Fielder is the wild-type wheat variety. DETAILED DESCRIPTION

[0031] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified. The reagents and materials described are commercially available unless otherwise specified. The percentages in the following examples are percentages by mass unless otherwise specified.

[0032] Example 1

[0033] This embodiment provides HASP 97 Acquisition of genes.

[0034] First, qRT-PCR was used to determine the function of effector protein HASP 97 in the interaction between wheat and stripe rust. TaEF1-α Real-time quantitative PCR was performed using specific primers encoding the wheat stripe rust effector protein HASP 97 as an internal reference to determine the expression level of the effector protein HASP 97 at different time points after stripe rust infection. qRT-PCR was also used to detect the induced expression of the effector protein in wild-type wheat Fielder inoculated with stripe rust CYR23 (non-compatible) and stripe rust CYR31 (compatible) at different time points (6 h, 12 h, 24 h, 48 h, 96 h, 120 h, and 180 h of stripe rust infection).

[0035] HASP 97 Quantitation primers:

[0036] Forward primer: HASP 97 -qRT-F: CTCGGCCCGTACTTTGAACT;

[0037] Reverse primer: HASP 97 -qRT-R:TTCGACGTTCTTCCCACCTG.

[0038] Internal Reference TaEF1-α Primers:

[0039] Forward primer: TaEF1α-F :TGGTGTCATCAAGCCTGGTATGGT;

[0040] Reverse primer: TaEF1α -R:ACTCATGGTGCATCTCAACGGACT.

[0041] The test results are as follows Picture 1 As shown, the results show HASP 97 The gene expression level reached the highest level 24 h after infection in the affinity system (inoculated with CYR31), indicating that the expression of the effector protein HASP 97 was induced by wheat stripe rust and that the effector protein HASP 97 may be involved in wheat resistance to stripe rust.

[0042] Example 2

[0043] This embodiment provides a method for breeding wheat varieties resistant to stripe rust.

[0044] Build includes HASP 97 A gene silencing expression vector of a gene; wheat immature embryos are transformed using an Agrobacterium-mediated genetic transformation method to obtain a gene silencing wheat material containing an effector protein HASP 97 gene fragment.

[0045] S101, obtain HASP 97 Gene silencing of wheat, to obtain HASP 97 Molecular detection of gene-silenced wheat;

[0046] S102, the T1 generation gene silenced plants were inoculated with the stripe rust epidemic race CYR32, and the resistance of the transgenic plants to the stripe rust epidemic race was identified.

[0047] Gene silencing was performed in Line 3 and Line 7 using transgenic technology. HASP 97 get HASP 97 Transgenic Line3 and Line7 plants were detected using PCR technology HASP 97 The transgenic positive plants were detected by 1% agarose gel electrophoresis. Picture 2 As shown, Picture 2 HASP 97 is the gene-silenced plant strain; M is DNA Marker; WT is the wild type; Plasmid is the plasmid positive control group; H2O is the water control group. HASP 97 Both gene silenced Line3 and Line7 plants expressed HASP 97 , no trace of β-glucan was detected in wild-type plants or water. HASP 97 .

[0048] Obtained HASP 97 Gene silenced plants inoculated with the compatible strain CYR32 showed enhanced disease resistance in wheat, indicating HASP 97 It plays a negative regulatory role in the interaction between wheat and stripe rust.

[0049] The gene silenced plants were inoculated with CYR32 and phenotypic identification was performed. HASP 97 Gene silenced plants showed enhanced resistance to stripe rust CYR32. The expression vector map is shown in Picture 3 shown.

[0050] Forward primer: HASP 97 -nucleotide-F:CTCGGCCCGTACTTTGAACT;

[0051] Reverse primer: HASP 97 -Nucleotide-R:TTCGACGTTCTTCCCACCTG.

[0052] The embodiment of the present invention provides a wheat stripe rust effector protein gene HASP 97 Application in wheat stripe rust resistance improvement, gene silenced plants obtained by Agrobacterium-mediated genetic transformation were tested by PCR, wild type "Fielder" and HASP 97 The gene silenced plant positive strains were inoculated with the main epidemic race CYR32 of stripe rust. 14 days after inoculation, Picture 4 As shown, HASP 97 The gene silenced plants produced significantly less spores than the wild-type plants. HASP 97 Gene-silenced plants showed enhanced resistance to stripe rust.

[0053] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions 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 scope of protection determined by the present invention.

Claims

1. Effector Protein HASP 97 The application of the gene in improving disease resistance of wheat is characterized in that: The effector protein HASP 97 The nucleotide sequence of the gene is shown in SEQ ID NO: 2; Silencing the effector protein HASP 97 Genes that enhance wheat's resistance to stripe rust.

2. The use according to claim 1, characterized in that Silencing the effector protein HASP 97 Gene that reduces sporulation of stripe rust.

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