Method for regulating and controlling soft rot resistance of sweet potatoes by utilizing IbHD2C gene and application of method

Abstract

The invention discloses a method for regulating and controlling soft rot resistance of sweet potatoes by utilizing an IbHD2C gene and application of the method, and belongs to the technical field of gene engineering. The invention discloses an application of an IbHD2C gene (the nucleotide sequence is shown as SEQ ID NO.3) and an encoding protein (the amino acid sequence is shown as SEQ ID NO.4) thereof in regulation and control of soft rot resistance. Experiments prove that the resistance of sweet potatoes to soft rot can be remarkably improved by interfering the IbHD2C gene, and the resistance is reduced by overexpressing the gene. The invention provides a new target for disease-resistant breeding of sweet potatoes, solves the problems of dependence on chemical agents and lack of disease-resistant varieties in the prior art, and has the advantages of high efficiency, environmental protection and sustainability.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for regulating sweet potato soft rot resistance by utilizing the IbHD2C gene and an application thereof. Background Art

[0002] Sweet potatoes are high-yielding, highly adaptable to the environment, and contain a large amount of starch, making them not only an important food but also a valuable feed, industrial raw material, and alternative energy source. They are an excellent source of nutrients, including vitamins, potassium, iron, calcium, and minerals, and are highly valued for their medicinal properties, including anti-cancer, anti-diabetic, and anti-inflammatory properties.

[0003] Soft rot is a common disease of sweet potatoes during storage, primarily caused by Rhizopus stolonifer. The fungus invades through tuber wounds and root pores, secreting a variety of enzymes that rapidly break down the gelatinous layer and other components of the tuber cells, causing the sweet potato tissue to disintegrate and rot. This disease can spread rapidly, often causing entire sweet potatoes to rot in the cellar, resulting in severe economic losses. Therefore, breeding disease-resistant sweet potato varieties has become a key measure to combat this disease.

[0004] Histone deacetylation is a key epigenetic modification that plays a crucial role in regulating gene expression. Histone deacetylases (HDACs) have been shown to be involved in plant disease resistance. For example, in rice, OsHDA706 deacetylates OsLOX14 and enhances its stability, leading to JA accumulation and enhanced resistance to rice stripe virus. In wheat, HOS15 can interact with HDA6 to regulate defense responses against powdery mildew. However, the role and regulatory relationship of HDACs in the interaction between sweet potato and pathogenic fungi have not been reported. Summary of the Invention

[0005] The present invention aims to provide a method and application for regulating sweet potato soft rot resistance using the IbHD2C gene to address the problems of the prior art. The present invention effectively controls sweet potato soft rot resistance by regulating the expression of the IbHD2C gene.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides applications of IbHD2C protein, including any of the following:

[0008] (1) Application in regulating sweet potato soft rot resistance;

[0009] (2) Application in the preparation of products for regulating sweet potato soft rot;

[0010] (3) Application in cultivating sweet potatoes resistant to soft rot;

[0011] The amino acid sequence of the IbHD2C protein is shown in SEQ ID NO.4.

[0012] The amino acid sequence of the IbHD2C protein is shown in SEQ ID NO. 4, or a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the IbERF114 protein as shown in SEQ ID NO. 4, or a protein having the same function as the amino acid sequence shown in SEQ ID NO. 4 by substitution and / or deletion and / or addition of one or more amino acid residues.

[0013] The present invention also provides an application of the gene IbHD2C encoding the IbHD2C protein, wherein the application includes any of the following:

[0014] (1) Application in regulating sweet potato soft rot resistance;

[0015] (2) Application in the preparation of products for regulating sweet potato soft rot;

[0016] (3) Application in cultivating sweet potatoes resistant to soft rot;

[0017] The nucleotide sequence encoding gene IbHD2C is shown in SEQ ID NO.3.

[0018] The present invention also provides an application of a recombinant vector containing the gene encoding IbHD2C, wherein the application includes any of the following:

[0019] (1) Application in regulating sweet potato soft rot resistance;

[0020] (2) Application in the preparation of products for regulating sweet potato soft rot;

[0021] (3) Application in cultivating sweet potatoes resistant to soft rot;

[0022] The nucleotide sequence encoding gene IbHD2C is shown in SEQ ID NO.3.

[0023] The present invention also provides applications of host bacteria containing the recombinant vector, wherein the applications include any of the following:

[0024] (1) Application in regulating sweet potato soft rot resistance;

[0025] (2) Application in the preparation of products for regulating sweet potato soft rot;

[0026] (3) Application in cultivating sweet potatoes resistant to soft rot;

[0027] The recombinant vector is a vector that integrates the coding gene IbHD2C into the genome. The nucleotide sequence of the coding gene IbHD2C is shown in SEQ ID NO.3.

[0028] Furthermore, the regulation is negative regulation.

[0029] The present invention also provides a method for regulating the soft rot resistance of sweet potato, comprising regulating the expression level of the IbHD2C gene in the sweet potato, wherein the nucleotide sequence of the IbHD2C gene is shown in SEQ ID NO.3.

[0030] Furthermore, the regulation is to enhance the resistance of sweet potato to soft rot by interfering with the expression of IbHD2C gene.

[0031] Furthermore, the regulation is to reduce the resistance of sweet potato to soft rot by overexpressing the IbHD2C gene.

[0032] The present invention also provides a method for improving the soft rot resistance of sweet potatoes, comprising interfering with the expression of the IbHD2C gene in the sweet potatoes, wherein the nucleotide sequence of the IbHD2C gene is shown in SEQ ID NO.3.

[0033] The present invention also provides a method for cultivating soft rot-resistant sweet potatoes, comprising the steps of interfering with the IbHD2C gene in the sweet potatoes, reducing the expression level of the IbHD2C gene, and obtaining the soft rot-resistant sweet potatoes; the nucleotide sequence of the IbHD2C gene is shown in SEQ ID NO.3.

[0034] The present invention discloses the following technical effects:

[0035] The IbHD2C gene provided by the present invention encodes a histone deacetylase. This gene was introduced into sweet potatoes to obtain transgenic sweet potato plants that overexpress the IbHD2C gene. The transgenic tubers were then tested for resistance to soft rot. It was found that compared with wild-type tubers, tubers overexpressing IbHD2C had significantly reduced resistance to soft rot, while tubers with IbHD2C knockout significantly improved disease resistance. The IbHD2C protein and its encoding gene provided by the present invention have important theoretical significance and application value in the study of sweet potato soft rot resistance. This invention provides a new target for disease-resistant sweet potato breeding, solving the existing problems of reliance on chemical agents for soft rot prevention and control and the lack of disease-resistant varieties. It has the advantages of high efficiency, environmental protection, and sustainability. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] 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. 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.

[0037] Figure 1 The following are the results of the acquisition and identification of transgenic plants: (a) is a stem segment of Yanshu 25 infected with Agrobacterium rhizogenes; (b) is an infected Yanshu 25 plant transplanted into the field; (c) is an IbHD2C transgenic plant grown in the field for 60 days; (d) is an IbHD2C transgenic root tuber grown for 120 days; (e) is the electrophoresis detection result of PCR amplification products of IbHD2C-overexpressing plants; (f) is the electrophoresis detection result of PCR amplification products of IbHD2C-interference plants; (g) is a statistical graph of IbHD2C expression levels in overexpressing and interference plants;

[0038] Figure 2 This is the identification of potato chip resistance to soft rot; (a) is the diseased image of potato chips after inoculation with soft rot bacteria; (b) is the statistical graph of diseased diameter. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] The experimental methods in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0045] Example 1 Obtaining and functional verification of proteins and their encoding genes related to sweet potato soft rot

[0046] 1. Obtaining proteins and their encoding genes related to sweet potato soft rot

[0047] Experimental materials: The sweet potato variety Nongdabai (NDB) was used as the experimental material.

[0048] 1. Extraction of total RNA from sweet potato: Grind 1 g of young leaves of the sweet potato line NDB into powder in liquid nitrogen and add the powder to a 2 mL centrifuge tube. Extract total RNA from the sweet potato using the Trizon method and reverse transcribe the first-strand cDNA using the PrimeScript™ RT reagent Kit with gDNAEraser.

[0049] 2. Using the existing transcriptome data in the laboratory, we screened the differentially expressed gene IbHD2C and compared it with the Sweetpotato Garden library to obtain an EST sequence. Based on the nucleotide sequence of the EST sequence, we designed and synthesized primers IbHD2C-F and IbHD2C-R. The sequence is:

[0050] IbHD2C-F: 5'-ATGGAGTTTTGGGGTGTTGAG-3' (SEQ ID NO. 1);

[0051] IbHD2C-R: 5'-CTTTCCAGAACTATGTTTAGCC-3' (SEQ ID NO. 2).

[0052] Using the cDNA obtained in step 1 as a template and primers IbHD2C-F and IbHD2C-R synthesized in step 2 as primers, PCR amplification was performed to obtain an approximately 867-bp PCR amplification product, which was then sequenced. Sequencing revealed the nucleotide sequence of the IbHD2C gene as shown in SEQ ID NO. 3, and the amino acid sequence of the encoded IbHD2C protein as shown in SEQ ID NO. 4.

[0053] SEQ ID NO.3:

[0054] ;

[0055] SEQ ID NO.4:

[0056] MEFWGVEVKSGEPFSVKPGDGMVLHLSQANLGEVKKLHGSESVCLFVNVDGRKLVLGTLISDKLPQQQFDLIFDKDFELSHNWKNGNVYFYGFKAQNPIADDDDDQESDSDEDSPLTFKAEPETDVKPEKQAGPEKSDVAKSSVS DKKVIIKEPHKDVKAEEDESSDEDMVSSDEDMDSSDGEDEDESDETPKKVESGKKRLAESAKKTPASDKNKKAKLETPQKTGGKKSSVHVATPYPLKQAKTAANKPNQSTPKSAGSHACKSCNRTFGSEGALDSHTKAKHSSGK.

[0057] 2. Application of IbHD2C protein in regulating sweet potato soft rot resistance

[0058] 1. Construction of plant expression vector

[0059] Based on the coding sequence of the sweet potato IbHD2C protein nucleotide, primer sequences were designed to amplify the complete coding sequence. The forward and reverse primers introduced Kpn I and Sal I restriction sites, respectively. The primer sequences are as follows:

[0060] IbHD2C-Kpn IF:

[0061] 5'-ACTCTTGACCATGGTGGTACCATGGAGTTTTGGGGTGTTGAG-3' (SEQ ID NO. 5) (the underlined portion is the KpnI restriction site);

[0062] IbHD2C-Sal IR:

[0063] 5'-GTCACCTGTAATTCA GTCGAC CTTTCCAGAACTATGTTTAGCC-3' (SEQ ID NO. 6) (the underlined portion is the Sal I restriction enzyme cleavage site).

[0064] Using the sequence shown in SEQ ID NO.3 as a template, after PCR amplification, the product was ligated into the pMD19-T vector, named pMD-IbHD2C vector, and M13-F / R sequencing was performed to ensure the correct reading frame and enzyme cleavage sites of the sweet potato IbHD2C protein nucleotides.

[0065] The pCAMBIA1300 vector was double-digested with Kpn I and Sal I to recover the large vector fragment. Simultaneously, a fragment of approximately 867 bp was amplified from the pMD-IbHD2C vector using primers IbHD2C-Kpn IF and IbHD2C-Sal IR. The recovered large vector fragment was then ligated with the 867 bp fragment to generate the target plasmid. The target plasmid was transformed into Escherichia coli DH5α and cultured at 37°C for 20 hours. The recombinant vector was analyzed by PCR, digested with enzymes, and verified by sequencing. Sequencing results revealed that the sequence shown in SEQ ID NO. 3 was inserted between the Kpn I and Sal I restriction sites of the pCAMBIA1300 vector, indicating that the recombinant vector was constructed correctly. The recombinant plasmid pCAMBIA1300-IbHD2C expressed the IbHD2C protein shown in SEQ ID NO. 4.

[0066] The recombinant plasmid pCAMBIA1300-IbHD2C has an expression cassette, and the nucleotide sequence of the expression cassette includes a CaMV35S promoter, a gene encoding IbHD2C protein and a NOS terminator.

[0067] 2. Construction of plant interference vectors

[0068] Based on the coding sequence of the sweet potato IbHD2C protein nucleotide, primer sequences for amplifying the interference fragment were designed. The primer sequences are shown in SEQ ID NO.7-SEQ ID NO.10:

[0069] IbHD2C-5941-UF-Xho I:

[0070] 5′-TTTGGAGAGGACACGCTCGAG ATGGAGTTTTGGGGTGTTGAG-3′ (SEQ ID NO. 7);

[0071] IbHD2C-5941-UR-Swa I:

[0072] 5′-AAGAAATTCTTACACATTTAAATTGTTGCTGGGGAAGCTTATC-3′ (SEQ ID NO. 8);

[0073] IbHD2C-5941-DF-BamH I:

[0074] 5′-AATTTGCAGGTATTTGGATCCTGTTGCTGGGGAAGCTTATC-3′ (SEQ ID NO. 9);

[0075] IbHD2C-5941-DR-Xba I:

[0076] 5'-GGTCTTAATTAACTCTCTAGAATGGAGTTTTGGGGTGTTGAG-3' (SEQ ID NO. 10).

[0077] Plasmid pFGC-5941 was double-digested with Xho I and Swa I, then ligated with the forward target sequence of IbHD2C, a homology arm of pFGC-5941 containing both Xho I and Swa I restriction sites. This was then ligated with the forward target sequence of IbHD2C amplified using primers IbHD2C-5941-UF-Xho I and IbHD2C-5941-UR-Swa I. This recombinant cloning vector containing the IbHD2C forward fragment was transformed into competent E. coli DH5α cells and, after correct sequencing, was named IbHD2C-5941F. IbHD2C-5941F was then subjected to a double restriction enzyme digestion reaction with BamH I and Xba I, and ligated with the reverse target sequence of the homology arm IbHD2C of pFGC5941 containing the double restriction enzyme sites of BamH I and Xba I. It was then ligated with the reverse target sequence of IbHD2C amplified using primers IbHD2C-5941-DF-BamH I and IbHD2C-5941-DR-Xba I, and then transformed into Escherichia coli. Finally, a recombinant RNAi expression vector with an inverted repeat sequence was obtained, which was named pFGC-IbHD2C-5941 after correct sequencing.

[0078] 3. Transformation of plant expression vector into Agrobacterium

[0079] (1) Thaw the prepared Agrobacterium K599 competent cells on ice, add 2 μg of the extracted pCAMBIA1300-IbHD2C / pFGC-IbHD2C-5941 plasmid, gently tap the tube wall to mix, and incubate on ice for 10 min;

[0080] (2) Quick freezing in liquid nitrogen for 5 min, 37°C water bath for 10 min, and ice bath for 5 min;

[0081] (3) Add 600 μL of liquid LB medium and incubate at 28°C, 200 rpm for 5 h;

[0082] (4) Spread 200 μL of bacterial solution on LB solid medium containing 100 μg / ml streptomycin and 100 μg / ml rifampicin;

[0083] (5) Culture in the dark at 28°C for 2 days. Take an appropriate amount of Agrobacterium and culture it in liquid LB medium for later use. This is to obtain Agrobacterium culture liquid introduced with the pCAMBIA1300-IbHD2C vector. The recombinant Agrobacterium was named K599 / pCAMBIA1300-IbHD2C and K599 / pFGC-IbHD2C-5941.

[0084] 4. Genetic transformation and regeneration of sweet potato

[0085] Using recombinant Agrobacterium infection of Yanshu 25 stem segments ( Figure 1 a), transplant the infected tobacco potato 25 to the field ( Figure 1 b), IbHD2C transgenic plants grown in the field for 60 days. Figure 1 As shown in c, the IbHD2C transgenic tobacco tubers harvested after 120 days of growth are as follows Figure 1 As shown in d, it can be seen that there is no significant difference between it and the wild type Yanshu 25.

[0086] Genomic DNA from leaves of the proposed transgenic plants was extracted using the CTAB method. PCR amplification was performed using the extracted genomic DNA as a template, water and wild-type plants as negative controls, and plasmid pCAMBIA1300-IbHD2C as a positive control, using primers IbHD2C-Kpn IF and IbHD2C-Sal IR to obtain a PCR amplification product. If the PCR amplification product contained a band of approximately 867 bp, the corresponding proposed transgenic sweet potato plant was a transgenic-positive sweet potato plant. PCR amplification was performed using primers IbHD2C-5941-UF-Xho I and 5941-1R (CTACCTTCCCACAATTCGTC (SEQ ID NO. 11)) to obtain a PCR amplification product. If the PCR amplification product contained a band of approximately 200 bp, the corresponding sweet potato plant was a transgenic sweet potato plant.

[0087] The results of electrophoresis detection amplification are shown in Figure 1 Zhongehe Figure 1 f in (lane M is Maker, lane W is negative control water; lane P is positive control (recombinant plasmid pCAMBIA1300-IbHD2C); lane WT is wild-type sweet potato plants; lanes OE-H1-OE-H9 are overexpressed sweet potato plants; lanes Ri-H1-Ri-H6 are interference sweet potato plants. Figure 1 Zhongehe Figure 1 As can be seen from the figure, the target bands were amplified in lanes OE-H1-OE-H9, Ri-H1-Ri-H6 and the positive control, indicating that the IbHD2C gene has been integrated into the sweet potato genome. Figure 1As can be seen in Figure g, the expression of the IbHD2C gene was significantly reduced in the interfered plants, indicating that the present invention successfully interfered with the IbHD2C gene.

[0088] 5. Inoculation and identification of soft rot in transgenic plants

[0089] Disease-free, healthy tubers from transgenic lines (OE-H2, OE-H3), interference lines (Ri-H4, Ri-H5), and wild-type tubers (WT) were washed and dried and then inoculated with the soft rot pathogen to identify their resistance to the disease. The specific inoculation method is as follows:

[0090] (1) Preparation of bacterial cells: 2-day-old soft rot pathogens were cultured on PDB medium. The surface hyphae were scraped off with an inoculating needle and placed in a flask containing 20 mL of PDB medium. A 0.5 cm diameter PDB solid medium disc was then added to the flask. The flask was shaken at 60 rpm for 2 h to allow a certain amount of bacterial cells to remain on the disc for later use.

[0091] (2) Preparation of potato tubers: Clean the potato tubers, dry them, cut them into slices about 1 cm thick, place them on a sterilized plate, and spray them with sterile water to keep them moist;

[0092] (3) Inoculation identification: Inoculate a disc in the middle of a potato chip. After inoculation, place the plate in a 28°C constant temperature incubator for 24 hours, spraying with sterile water once to keep it moist.

[0093] (4) Disease evaluation: Measure the disease diameter of potato chips 24 hours after inoculation.

[0094] The results are as follows Figure 2 As shown in the figure, the diameter of the diseased leaves in the overexpressing plants was significantly larger than that in the wild-type plants, while the diameter of the diseased leaves in the interfered plants was significantly smaller than that in the overexpressing plants, indicating that interfering with the IbHD2C gene in sweet potato can improve its resistance to soft rot. Therefore, the sweet potato soft rot-related protein IbHD2C and its encoding gene have important theoretical significance in regulating plant soft rot resistance.

[0095] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of IbHD2C protein, characterized in that: The application includes any of the following: (1) Application in regulating sweet potato soft rot resistance; (2) Application in the preparation of products for regulating sweet potato soft rot; (3) Application in cultivating sweet potatoes resistant to soft rot; The amino acid sequence of the IbHD2C protein is shown in SEQ ID NO.

4.

2. Use of the gene IbHD2C encoding the IbHD2C protein, characterized in that: The application includes any of the following: (1) Application in regulating sweet potato soft rot resistance; (2) Application in the preparation of products for regulating sweet potato soft rot; (3) Application in cultivating sweet potatoes resistant to soft rot; The nucleotide sequence encoding gene IbHD2C is shown in SEQ ID NO.

3.

3. Use of a recombinant vector containing the coding gene IbHD2C, characterized in that: The application includes any of the following: (1) Application in regulating sweet potato soft rot resistance; (2) Application in the preparation of products for regulating sweet potato soft rot; (3) Application in cultivating sweet potatoes resistant to soft rot; The nucleotide sequence encoding gene IbHD2C is shown in SEQ ID NO.

3.

4. The use of host bacteria containing recombinant vectors, characterized in that: The application includes any of the following: (1) Application in regulating sweet potato soft rot resistance; (2) Application in the preparation of products for regulating sweet potato soft rot; (3) Application in cultivating sweet potatoes resistant to soft rot; The recombinant vector is a vector that integrates the coding gene IbHD2C into the genome. The nucleotide sequence of the coding gene IbHD2C is shown in SEQ ID NO.

3.

5. The use according to any one of claims 1 to 4, characterized in that The regulation is negative regulation.

6. A method for regulating sweet potato soft rot resistance, characterized in that: The method comprises regulating the expression level of the IbHD2C gene in the sweet potato, wherein the nucleotide sequence of the IbHD2C gene is shown in SEQ ID NO.

3.

7. The method according to claim 6, characterized in that The regulation is to enhance the resistance of sweet potato to soft rot by interfering with the expression of IbHD2C gene.

8. The method according to claim 6, characterized in that The regulation is to reduce the resistance of sweet potato to soft rot by overexpressing the IbHD2C gene.

9. A method for improving the resistance of sweet potatoes to soft rot, characterized in that: The method comprises interfering with the expression of the IbHD2C gene in sweet potato, wherein the nucleotide sequence of the IbHD2C gene is shown in SEQ ID NO.

3.

10. A method for cultivating sweet potatoes resistant to soft rot, characterized in that: The method comprises the steps of interfering with the IbHD2C gene in the sweet potato, reducing the expression level of the IbHD2C gene, and obtaining the soft rot-resistant sweet potato; the nucleotide sequence of the IbHD2C gene is shown in SEQ ID NO.3.

Images