MsWRKY61 protein for improving resistance of valsa mali, gene and application of MsWRKY61 protein and gene

Abstract

The invention discloses an MsWRKY61 protein for improving resistance of valsa mali, a gene and application of the MsWRKY61 protein and the gene, and belongs to the technical field of gene engineering. The amino acid sequence of the MsWRKY61 protein is as shown in SEQ ID NO. 2, and the nucleotide sequence of the MsWRKY61 gene is as shown in SEQ ID NO. 1. According to the invention, the valsa mali resistant gene MsWRKY61 is identified, and the resistance of valsa mali can be remarkably enhanced by overexpressing the MsWRKY61 gene. According to the application disclosed by the invention, phenotypes of MsWRKY61 transgenic lines before and after germ infection treatment are analyzed to show that the MsWRKY61 overexpression transgenic lines have obvious valsa ceratosperma resistance characteristics for non-transgenic lines, so that the MsWRKY61 gene is a potential valsa ceratosperma resistance gene, and a new gene resource and a technical path are provided for cultivating disease-resistant apple varieties.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a MsWRKY61 protein, gene, and its application for enhancing resistance to apple rot pathogens. Background Technology

[0002] Apples are highly valued by consumers for their nutritional quality, unique flavor, and good storage properties. However, apple production is severely threatened by apple canker, a fungal pathogen caused by *Phyllostachys rubrum*, one of the most destructive diseases in apple cultivation worldwide. This disease leads to tree decline, branch necrosis, and even the death of the entire tree, causing significant economic losses. *Phyllostachys rubrum* primarily infects wound tissue. After invading the wound, the fungal hyphae grow intercellularly and intracellularly, colonizing all bark tissues, leading to severe tissue softening and necrosis. Such infections often result in the death of branches, large twigs, or the entire tree. Due to the perennial nature of apple trees and the extensive colonization of the pathogen in the phloem and xylem of the host, effectively controlling apple canker using agrochemicals remains challenging. Current management strategies mainly rely on the application of fungicides, physical removal of lesions, and pruning of weak or dead branches. However, the long-term widespread use of conventional synthetic fungicides has led to pesticide residues in crops, the evolution of fungicide resistance, and environmental safety issues. Therefore, there is an urgent need to identify natural compounds that can effectively alleviate this disease. Against this backdrop, genetic engineering is one of the most promising and practical strategies for achieving persistent control of apple rot pathogen infection.

[0003] Evidence suggests that transcription factors, such as ethylene response factors, WRKY, and MYB, play crucial roles in plant defense against pathogen invasion. Among these families, the WRKY family is one of the largest transcription factor groups in plants, forming a core component of complex signaling networks and precisely regulating a variety of physiological processes. WRKY transcription factors primarily participate in plant immune responses to biotic stresses by transcribing downstream target genes. For example, PtrWRKY73, a salicylic acid-induced poplar WRKY transcription factor, is involved in Arabidopsis disease resistance; GhWRKY15 is involved in cotton disease resistance and development. However, the specific WRKY transcription factors involved in apple rot disease remain to be investigated. Summary of the Invention

[0004] The purpose of this invention is to provide a MsWRKY61 protein, gene, and its application to improve resistance to apple rot pathogens, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] In a first aspect, the present invention provides a MsWRKY61 protein that enhances resistance to apple rot pathogens, the amino acid sequence of which is shown in SEQ ID NO.2.

[0007] Secondly, the present invention also provides a gene encoding the above-mentioned MsWRKY61 protein, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] Thirdly, the present invention also provides a gene expression cassette comprising the aforementioned gene.

[0009] Fourthly, the present invention also provides a recombinant expression vector comprising the above-mentioned gene expression cassette.

[0010] Fifthly, the present invention also provides a recombinant host cell comprising the above-described recombinant expression vector.

[0011] Preferably, the recombinant host cell is recombinant Agrobacterium.

[0012] In a sixth aspect, the present invention also provides the application of the above-mentioned gene, the above-mentioned gene expression cassette, the above-mentioned recombinant expression vector, or the above-mentioned recombinant host cell in improving resistance to apple rot pathogens.

[0013] In a seventh aspect, the present invention also provides a method for improving resistance to apple rot pathogens by overexpressing the MsWRKY61 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0014] Preferably, the method for overexpressing the MsWRKY61 gene includes the step of transforming the above-described recombinant expression vector into apple plants.

[0015] Preferably, the method for overexpressing the MsWRKY61 gene includes the step of transforming the above-described recombinant host cells into apple plants.

[0016] The present invention discloses the following technical effects:

[0017] This invention identified a gene, MsWRKY61, that resists apple rot pathogens. Overexpression of the MsWRKY61 gene significantly enhances resistance to these pathogens. Analysis of the phenotypes of MsWRKY61 transgenic lines before and after pathogen infection treatment showed that, compared to non-transgenic lines, MsWRKY61-overexpressing transgenic lines exhibited significant resistance to rot pathogens. This indicates that the MsWRKY61 gene is a potential source of resistance to apple rot pathogens, providing a new genetic resource and technical pathway for breeding disease-resistant apple varieties. This could promote the development of apple cultivation industries resistant to rot pathogens and overcome the limitations imposed by rot pathogens on high-yield apple production. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0019] Figure 1 The study evaluated the disease symptoms and resistance of the overexpression line and control plants after infection with the pathogen. Among them, A shows the quantitative results of MsWRKY61 gene expression in MsWRKY61 transgenic apples compared with the control; B shows the phenotypic results of different groups 3 days after inoculation with the pathogen; and C shows the statistical results of the infection area of ​​different groups after inoculation with the pathogen. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] The nucleotide sequence of the MsWRKY61 gene involved in this invention is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0026] SEQ ID NO.1:

[0027]

[0028] SEQ ID NO.2:

[0029] .

[0030] The wild apples of Xinjiang are preserved by the Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences.

[0031] Example 1: Screening of the MsWRKY61 gene, a regulatory factor against apple rot pathogens.

[0032] To investigate genes playing a key regulatory role against *A. erythroplasma gondii*, this invention employed metabolomics, transcriptomics, and functional analyses to explore the defense mechanisms of wild apples against this pathogen. Healthy branch segments (10 mm in diameter) from the same tree were rinsed with double-distilled water, disinfected in 70% ethanol, and then rinsed with sterile water. Holes were punctured in these disinfected branches, and 5 mm mycelial blocks were inoculated from the edges of *A. erythroplasma gondii* colonies cultured on PDA medium. All inoculated branches were cultured at 25°C in the dark under high humidity conditions. Bark samples from branches near the lesions were collected at 0, 1, 2, and 5 days post-inoculation, with each sample containing three biological replicates. All samples were collected for metabolomics, transcriptomics, and functional analyses. The results revealed that MsWRKY61 strongly responds to *A. erythroplasma gondii*.

[0033] Example 2: Overexpression of the MsWRKY61 gene enhances the resistance of apples to rot pathogens.

[0034] 1. Cloning and overexpression vector construction methods for the MsWRKY61 gene

[0035] Fresh apple tissue was ground in liquid nitrogen, and preheated CTAB extraction buffer (containing β-mercaptoethanol) was added. After treatment in a 65°C water bath, extraction was performed using chloroform-isoamyl alcohol. The resulting RNA was precipitated with LiCl and washed with 75% ethanol (volume fraction) to obtain total RNA. Using the All-in-One cDNA synthesis kit from TransGen Biotech, cDNA was obtained by reverse transcription using the qualified total RNA as a template.

[0036] Using cDNA as a template, MsWRKY61 fragments were obtained by PCR amplification with specific primers containing KpnI and XbaI restriction sites (and without stop codons) and Kangwei Century Biotechnology's Super Pfx Master Mix. After confirming the correct size of the amplified product by agarose gel electrophoresis, it was recovered using a gel recovery kit.

[0037] Primer sequence information is as follows:

[0038] MsWRKY61-pCAMBIA2300-F:

[0039] acggggggacgagctcggtaccATGCCAAAGAGGCCTTCAAAA (SEQ ID NO. 3);

[0040] MsWRKY61-pCAMBIA2300-R:

[0041] aggtcgactctagaggatccTCACAAAGATTCGTTCAAGTAGCTTGATG (SEQ ID NO. 4);

[0042] The pCAMBIA2300 vector was double-digested with KpnI and XbaI restriction endonucleases, and the vector backbone fragment was recovered after electrophoresis of the digestion products.

[0043] ClonExpress, using Novizan biotechnology ® The Ultra One-Step Cloning Kit was used to ligate the recovered MsWRKY61 fragment with the vector backbone at a ratio of 1:5. The ligation product was transformed into E. coli DH5α, and after kanamycin resistance selection, restriction enzyme digestion verification, and sequencing confirmation, the recombinant expression vector was obtained. The recombinant plasmid was introduced into Agrobacterium tumefaciens EHA105 competent cells, and positive strains were obtained after resistance selection and PCR verification.

[0044] 2. Transgenic apple tissue culture seedlings overexpressing MsWRKY61 were obtained through transient transformation.

[0045] Positive strains were inoculated into YEP medium containing 50 mg / L kanamycin and 25 mg / L rifampin and cultured at 28°C until OD500. 600 The OD value was set at 0.8; the bacterial cells were collected by centrifugation and resuspended in MS liquid medium containing 50 g / L sucrose, 150 μM acetylsyringone, 5 mM CaCl2 and 0.01% Tween 20, and the OD value was adjusted. 600 Bring the solution to 1.0 and let it stand for 30 minutes. Take healthy 1-month-old apple tissue culture seedlings, immerse them in the bacterial suspension, and place them in a vacuum desiccator for 15 minutes of vacuum permeation. After removal, rinse them three times with distilled water, blot dry with sterile filter paper, and return them to the original MS solid medium. Incubate at 25°C under 16-hour light / 8-hour dark conditions. This yields transgenic apple tissue culture seedlings overexpressing MsWRKY61.

[0046] 3. Phenotypic analysis of MsWRKY61-overexpressing transgenic apple tissue culture seedlings

[0047] The pathogen Valsa mali used for infection was inoculated onto PDA medium and cultured in the dark at 25°C for 5 days. The experimental group consisted of transgenic apple tissue culture seedlings overexpressing MsWRKY61 (MsWRKY61-L1, MsWRKY61-L2), while the control group consisted of apple tissue culture seedlings overexpressing the empty vector (ET-EV).

[0048] Leaves from both the experimental and control groups were cut off with scissors and placed in petri dishes lined with filter paper. Sterile water was added to the filter paper for moisture retention, and the petioles were wrapped with damp cotton wool. Then, symmetrically along the midrib, punctures were made on both sides of the leaf using a syringe needle. Subsequently, holes were punched in the leaves with a 5mm diameter punch, and the fungal cake was applied to the puncture wounds. The petri dishes were then sealed with sealing film and incubated in the dark at 25°C for 3 days.

[0049] The results showed that the growth of transgenic apple seedlings overexpressing MsWRKY61 was significantly better than that of transgenic apple seedlings overexpressing empty vector. Three days after inoculation with pathogens, the pathogen infection area of ​​transgenic apple seedlings overexpressing MsWRKY61 was lower than that of the control group. Figure 1 This demonstrates that overexpression of MsWRKY61 significantly enhances the resistance of apples to rot pathogens.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A MsWRKY61 protein that enhances resistance to Valsa mali, characterized in that, The amino acid sequence of the MsWRKY61 protein is shown as SEQ ID NO.

2.

2. A gene encoding the MsWRKY61 protein according to claim 1, characterized in that, The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

3. A gene expression cassette, characterized in that, The gene of claim 2.

4. A recombinant expression vector, characterized in that, The gene expression cassette of claim 3.

5. A recombinant host cell, characterized in that, The recombinant expression vector of claim 4.

6. The recombinant host cell of claim 5, wherein, The recombinant host cell is a recombinant Agrobacterium.

7. Use of the gene of claim 2, the gene expression cassette of claim 3, the recombinant expression vector of claim 4, or the recombinant host cell of any one of claims 5-6 in improving resistance of V. mali to Valsa mali.

8. A method for improving resistance to apple rot pathogens, characterized in that, The resistance of V. mali to Valsa mali is improved by overexpressing the MsWRKY61 gene, the nucleotide sequence of which is shown as SEQ ID NO.

1.

9. The method of claim 8, wherein, The method for overexpressing the MsWRKY61 gene comprises the step of transforming the recombinant expression vector of claim 4 into an apple plant.

10. The method of claim 8, wherein, The method for overexpressing the MsWRKY61 gene comprises the step of transforming the recombinant host cell of any one of claims 5-6 into an apple plant.

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