A method for improving resistance to sweet potato soft rot

Abstract

The present invention discloses a method for improving the resistance of sweet potato to soft rot, belonging to the technical field of plant genetic engineering. The present invention provides the use of the IbJAZ10 protein in any of the following: (1) use in regulating the soft rot of sweet potato; (2) use in preparing a product for regulating the soft rot of sweet potato; (3) use in breeding sweet potato varieties resistant to soft rot; (4) use in cultivating sweet potato varieties resistant to soft rot; the amino acid sequence of the IbJAZ10 protein is shown in SEQ ID NO.3. Experimental verification shows that the disease resistance of plants interfered with by the IbJAZ10 gene is significantly improved compared with the wild type. The IbJAZ10 protein and its encoding gene provided by the present invention have important theoretical significance and application value in the study of resistance to soft rot of sweet potato.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, in particular to a method for improving the resistance of sweet potato to soft rot. 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 the plant through 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, developing new, disease-resistant sweet potato varieties is a key measure to combat this disease. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for improving the resistance of sweet potato to soft rot, so as to solve the problems existing in the above-mentioned prior art. Experimental verification shows that IbJAZ10 protein negatively regulates sweet potato soft rot, which is of great significance for cultivating sweet potato resistant to soft rot.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides use of the IbJAZ10 protein in any of the following:

[0007] (1) Application in regulating sweet potato soft rot;

[0008] (2) Application in the preparation of products for regulating sweet potato soft rot;

[0009] (3) Application in breeding sweet potato varieties resistant to soft rot;

[0010] (4) Application in cultivating sweet potatoes resistant to soft rot;

[0011] The amino acid sequence of the IbJAZ10 protein is shown in SEQ ID NO. 3.

[0012] The present invention also provides the use of the gene IbJAZ10 encoding the IbJAZ10 protein in any of the following:

[0013] (1) Application in regulating sweet potato soft rot;

[0014] (2) Application in the preparation of products for regulating sweet potato soft rot;

[0015] (3) Application in breeding sweet potato varieties resistant to soft rot;

[0016] (4) Application in cultivating sweet potatoes resistant to soft rot;

[0017] The nucleotide sequence encoding the gene IbJAZ10 is shown in SEQ ID NO.2.

[0018] The present invention also provides the use of a recombinant vector containing the encoding gene IbJAZ10 in any of the following:

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

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

[0021] (3) Application in breeding sweet potato varieties resistant to soft rot;

[0022] (4) Application in cultivating sweet potatoes resistant to soft rot;

[0023] The nucleotide sequence encoding the gene IbJAZ10 is shown in SEQ ID NO.2.

[0024] The present invention also provides the use of a host bacterium containing a recombinant vector in any of the following:

[0025] (1) Application in regulating sweet potato soft rot;

[0026] (2) Application in the preparation of products for regulating sweet potato soft rot;

[0027] (3) Application in breeding sweet potato varieties resistant to soft rot;

[0028] (4) Application in cultivating sweet potatoes resistant to soft rot;

[0029] The recombinant vector is a vector that integrates the coding gene IbJAZ10 into the genome, and the nucleotide sequence of the coding gene IbJAZ10 is shown in SEQ ID NO.2.

[0030] Preferably, the regulation is negative regulation.

[0031] The present invention also provides a method for controlling sweet potato soft rot, comprising any one of the following methods:

[0032] (1) Improving the resistance of sweet potato to soft rot by interfering with the IbJAZ10 gene in sweet potato;

[0033] (2) reducing the resistance of sweet potatoes to soft rot by overexpressing the IbJAZ10 gene in sweet potatoes;

[0034] The nucleotide sequence of the IbJAZ10 gene is shown in SEQ ID NO.2.

[0035] The present invention also provides a method for cultivating a sweet potato resistant to soft rot, comprising the steps of interfering with the IbJAZ10 gene in the sweet potato, reducing the expression level of the IbJAZ10 gene, and obtaining the sweet potato resistant to soft rot;

[0036] The nucleotide sequence of the IbJAZ10 gene is shown in SEQ ID NO.2.

[0037] The present invention discloses the following technical effects:

[0038] Experimental verification shows that the present invention introduces the IbJAZ10 gene into sweet potato to obtain transgenic sweet potato plants that overexpress the IbJAZ10 gene. The tubers harvested from the transgenic plants are subjected to soft rot resistance identification, and it is found that compared with the wild type, the transgenic plants have significantly reduced resistance to soft rot; while compared with the wild type, the disease resistance of the IbJAZ10 gene-interfered plants is significantly improved. The IbJAZ10 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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.

[0040] Figure 1 Obtaining and identifying transgenic plants; A: Callus cultured on MS solid medium containing 2 mg / L 2,4-D; B: Embryogenic suspension cells cultured on MS liquid medium containing 2 mg / L 2,4-D; C: Embryogenic callus cultured on MS solid medium containing 300 mg / L CCS and 2 mg / L 2,4-D; D: Pseudo-transgenic plants regenerated on MS solid medium containing 1 mg / L ABA; E: Transgenic IbJAZ10 sweet potato plants subcultured on MS solid medium, the left picture shows the overexpression plant, and the right picture shows the interference plant; FG: Phenotypes of leaves (F) and sweet potato tubers (G) of overexpression and interference plants; HI: Gene electrophoresis results of overexpression and interference plants; J: Quantitative statistical graph of H and I;

[0041] Figure 2 Identification of potato chips' resistance to soft rot; A: Diseased potato chips after inoculation with soft rot pathogen; B: Statistical graph of diseased diameter. DETAILED DESCRIPTION

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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 illustrative only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The sweet potato varieties Shangshu 19 and Lizixiang used in the present invention are provided by the Key Laboratory of Sweet Potato Biology and Biotechnology of the Ministry of Agriculture and Rural Affairs of China Agricultural University, and the soft rot fungus used is Rhizopus stolonifer, which is also provided by the Key Laboratory of Sweet Potato Biology and Biotechnology of the Ministry of Agriculture and Rural Affairs of China Agricultural University.

[0048] Example 1

[0049] 1. Obtaining proteins and their encoding genes related to sweet potato soft rot

[0050] 1. Extraction of Total RNA from Sweet Potato

[0051] 1 g of young leaves of the sweet potato line Shangshu 19 were ground into powder in liquid nitrogen and added to a 2 mL centrifuge tube. Total RNA of the sweet potato was extracted using the Trizon method, and the first-strand cDNA was reverse transcribed using the PrimeScriptTMRT reagent Kit with gDNA Eraser kit.

[0052] 2. Screening genes

[0053] Using the previously studied sweet potato stress-resistance gene IbBBX24 (reference patent CN109306000A - Stress-Resistance-Related Protein IbBBX24, Its Encoding Gene, and Applications), we screened the interacting gene IbJAZ10 using a sweet potato yeast two-hybrid library. This gene was then aligned with the Sweetpotato Garden library to obtain an EST sequence (nucleotide sequence shown in SEQ ID NO. 1). Based on the EST sequence, we designed and synthesized primers IbJAZ10-F and IbJAZ10-R, the sequences of which are shown below.

[0054] IbJAZ10-F: 5′-ATGTCGAAACTGGATTCCG-3′ (SEQ ID NO.4);

[0055] IbJAZ10-R: 5'-CTAGGCTTGGCTCCCTAGAGC-3' (SEQ ID NO. 5).

[0056] 3. Synthetic genes

[0057] Using the cDNA obtained in step 1 as a template and IbJAZ10-F and IbJAZ10-R designed in step 2 as primers, PCR amplification was performed to obtain a PCR amplification fragment product of approximately 570 bp and sequenced.

[0058] The nucleotide sequence of the PCR amplification product is shown in SEQ ID NO.2. The gene shown in the sequence is named IbJAZ10 gene, the protein encoded by it is named IbJAZ10 protein, and its amino acid sequence is shown in SEQ ID NO.3.

[0059] SEQ ID NO.1 (EST sequence):

[0060] ATGTCGAAACTGGATTCCGCCGTTGAGCTCGATTTCTTCGGCATGGAGAATCAGGCCGCCGCCGCTAAGCCTCCACAGGCTCCCAAGCTCTTCGAGCGCCGCCGAAGCTTCCGCGATATTCAGCATGCGATTTCGAAGATCAATCCGGAGGTCGTGAAGAACGTGATTGCTTCCGCCTCTGCGAATTCGAAGGAGGAGCCGCGTCTCTTCCCGGCATTGCCTGTTTATGCGCCAGCTTCAAGGACTACTACTGTGTCCTCCGCCGAGAACGGCGGTGGCGAAACGGCGCCGTTGACTATCTTCTACGACGGAACCGTTTCCGTTTTTGACGTCCGCCGTCTCGAGGCGGAGAAAATTATCAAGCTTATTCAACAAGAAAGCCTCTTTAAAACTGCTGACTCGGCCGATTCCAACCTCGCAAATACCGATGGAGATCTGCCATTACAGAGGAGGAAATCTCTGCAAAGGTTTCTGGAAAAGCGCAGGTCAAGATTGGTTATGGTGTCACCTTATGGTTCTCCGTCTGAGTACGCAGCCTCAGGTGACACTGCTCTAGGGAGCCAAGCCTAG;

[0061] SEQ ID NO.2 (IbJAZ10 gene):

[0062] ATGTCGAAACTGGATTCCGCCGTTGAGCTCGATTTCTTCGGCATGGAGAATCAGGCCGCCGCTAAGCCTCCACAGGCTCCCAAGCTCTTCGAGCGCCGCCGAAGCTTCCGCGATATTCAGCATGCGATTTCGAAGATCAATCTGGAGGTCGTGAAGAACGTGATTGCTTCCGCCTCTGCGAATTCGAAGGAGGAGCCGCGTCTCTTCCCGGCATTGCCTGTTTATGCGCCAGCTTCAAGGACTACTACTGTGTCCTCCGCCGAGAACGGCGGTGGCGAAACGGCGCCGTTGACTATCTTCTACGACGGAACCGTTTCCGTTTTTGACGTCCGCCGTCTCGAGGCGGAGAAAATTATCAAGCTTATTCAACAAGAAAGCCTCTTTAAAACTGCTGACTCGGCCGATTCCAACCTCGCAAATACCGATGGAGATCTGCCATTACAGAGGAGGAAATCTCTGCAAAGGTTTCTGGAAAAGCGCAGGTCAAGATTGGTTATGGTGTCACCTTATGGTTCTCCGTCTGAGTACGCAGTCTCAGGCGACATTGCTCTAGGGAGCCAAGCCTAG;

[0063] SEQ ID NO.3 (IbJAZ10 protein):

[0064] MSKLDSAVELDFFGMENQAAAKPPQAPKLFERRRSFRDIQHAISKINLEVVKNVIASASANSKEEPRLFPALPVYAPASRTTTVSSAENGGGETAPLTIFYDGTVSVFDVRRLEAEKIIKLIQQESLFKTADSADSNLANTDGDLPLQRRKSLQRFLEKRRSRLVMVSPYGSPSEYAVSGDIALGSQA。

[0065] II. Application of IbJAZ10 protein in regulating sweet potato soft rot resistance

[0066] 1. Construction of plant expression vector

[0067] Based on the coding sequence of the sweet potato IbJAZ10 protein nucleotide, primer sequences were designed to amplify the complete coding sequence. The forward and reverse primers introduced BglII and PmlI restriction sites, respectively. The primer sequences are as follows:

[0068] IbJAZ10-FF-BglII: 5′-ACTCTTGACCATGGT AGATCT ATGTCGAAACTGGATTCCG-3′ (SEQ ID NO. 6, the underlined portion is the BglII restriction site);

[0069] IbJAZ10-RR-PmlI:5′-GTCACCTGTAATTCA CACGTG CTAGGCTTGGCTCCCTAGAGC-3′ (SEQ ID NO. 7, the underlined part is the PmlI restriction site);

[0070] Using the sequence shown in SEQ ID NO.2 as a template, after PCR amplification, the product was ligated into the pMD19-T vector, named pMD-IbJAZ10 vector, and M13-F / R sequencing was performed to ensure the correct reading frame and restriction enzyme cleavage sites of the sweet potato IbJAZ10 protein nucleotides.

[0071] The pCAMBIA1300 vector was double-digested with HindIII and EcoRI to recover the large vector fragment. The pBI121 vector was double-digested with restriction endonucleases HindIII and EcoRI to recover the fragment containing the gusA gene. The recovered large vector fragment was ligated with the fragment containing the gusA gene to obtain the recombinant vector pCBGUS.

[0072] The recombinant vector pCBGUS was digested with BglII and PmlI to recover the large vector fragment. Simultaneously, a fragment of approximately 570 bp was amplified from the pMD-IbJAZ10 vector using primers IbJAZ10-FF and IbJAZ10-RR. The recovered large vector fragment was then ligated with the approximately 570 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 then analyzed by PCR, digested with enzymes, and verified by sequencing. Sequencing results revealed that the sequence shown in SEQ ID NO. 2 was inserted between the BglII and PmlI restriction sites of the pCBGUS vector, indicating that the recombinant vector was constructed correctly and that the recombinant plasmid pCAMBIA1300-IbJAZ10 expressed the IbJAZ10 protein.

[0073] The recombinant plasmid pCAMBIA1300-IbJAZ10 has an expression cassette, the nucleotide sequence of which includes a CaMV35S promoter, a gene encoding IbJAZ10 protein and a NOS terminator.

[0074] 2. Construction of plant interference vectors

[0075] Based on the coding sequence of the sweet potato IbJAZ10 protein nucleotide, primer sequences for amplifying the interference fragment were designed. The primer sequences are as follows:

[0076] IbJAZ10-5941-UF (Xho I, SEQ ID NO.8):

[0077] 5′-TTTGGAGAGGACACGCTCGAGCTTCCGCGATATTCAGCATG-3′;

[0078] IbJAZ10-5941-UR (Swa I, SEQ ID NO.9):

[0079] 5′-AAGAAATTCTTACACATTTAAATTAGAAGATAGTCAACGGCGCC-3′;

[0080] IbJAZ10-5941-DF (BamH I, SEQ ID NO. 10):

[0081] 5′-AATTTGCAGGTATTTGGATCCTAGAAGATAGTCAACGGCGCC-3′;

[0082] IbJAZ10-5941-DR (Xba I, SEQ ID NO. 11):

[0083] 5′-GGTCTTAATTAACTCTCTAGACTTCCGCGATATTCAGCATG-3′.

[0084] Plasmid pFGC-5941 was digested with Xho I and Swa I, then ligated with the forward target sequence of the pFGC-5941 homology arm IbJAZ10, which contains both Xho I and Swa I restriction sites. The resulting vector was then transformed into competent E. coli DH5α cells to generate a recombinant cloning vector containing the IbJAZ10 forward fragment. After correct sequencing, the vector was named IbJAZ10-5941F. IbJAZ10-5941F was then digested with BamH I and Xba I, then ligated with the reverse target sequence of the pFGC5941 homology arm IbJAZ10, which contains both BamH I and Xba I restriction sites. The resulting vector was then transformed into E. coli to generate a recombinant RNAi expression vector containing the inverted repeat sequence. After correct sequencing, the vector was named pFGC-IbJAZ10-5941.

[0085] 3. Transformation of Agrobacterium

[0086] (1) Thaw the prepared Agrobacterium EHA105 competent cells on ice, add 2 μg of the extracted pCAMBIA1300-IbJAZ10 / pFGC-IbJAZ10-5941 plasmid, gently tap the tube wall to mix, and incubate on ice for 10 min;

[0087] (2) Quick freezing in liquid nitrogen for 5 min, 37°C water bath for 10 min, and ice bath for 5 min;

[0088] (3) Add 600 μL of liquid LB medium and incubate at 28°C, 200 rpm for 5 h;

[0089] (4) Spread 200 μL of bacterial solution onto LB solid medium containing 100 μg / mL ampicillin and 100 μg / mL rifampicin;

[0090] (5) Incubate the cells in an inverted manner at 28°C in the dark for 2 days. Take an appropriate amount of Agrobacterium and culture it in liquid LB medium for later use. This will yield Agrobacterium culture solution containing the pCB-IbJAZ10 vector. The recombinant Agrobacterium will be named EHA105 / pCAMBIA1300-IbJAZ10 and EHA105 / pFGC-IbJAZ10-5941.

[0091] 4. Genetic transformation and regeneration of sweet potato

[0092] EHA105 / pCAMBIA1300-IbJAZ10 and EHA105 / pFGC-IbJAZ10-5941 were introduced into Ganshu Lizixiang using Agrobacterium-mediated transfection. The specific method is as follows:

[0093] (1) Gently rinse the stem tip retrieved from the greenhouse with distilled water to remove dust, residual leaves, etc. on the surface of the stem tip; remove the larger leaves on the explant, leaving the stem segment with the stem tip, preferably 1-1.5 cm in length; first soak and disinfect it with 70% ethanol solution for 30 seconds, then quickly transfer the stem tip to 2% sodium hypochlorite solution for disinfection for 5 minutes, gently shaking it during the process to ensure full contact between the stem tip and the sodium hypochlorite solution; after disinfection, pour out the waste liquid and rinse it with sterile water three times to remove any ethanol and sodium hypochlorite solution that may remain on the stem tip.

[0094] (2) Soak the cleaned stem segment with the stem tip in sterile water for the extraction of the stem tip meristem; use tweezers to pick up the stem tip soaked in sterile water and place it on a culture dish. Under a binocular microscope (20×), remove the leaves and leaf primordia surrounding the stem tip meristem from the outside to the inside (1-2 leaf primordia close to the meristem can be retained). Carefully cut off the exposed meristem (about 0.5 mm) and inoculate it into a 100 mL triangular flask containing 2.0 mg / L 2,4-D MS solid medium and culture in the dark to induce embryonic callus ( Figure 1 Middle A).

[0095] (3) After 6-8 weeks of dark culture at room temperature of 27±1℃, the shoot apical meristem will gradually form embryonic callus. The embryonic callus in good condition will be gently moved to MS liquid culture medium containing 2.0mg / L 2,4-D and broken into small cell clusters with tweezers. The cells will be cultured at 100rpm, 27±1℃, and 500Lx light (13h per day). After 8-12 weeks of culture, the suspension cells with good growth, rapid reproduction, and bright yellow color will be selected as the recipients of sweet potato genetic transformation for subsequent transformation work ( Figure 1 Middle B).

[0096] (4) Soak the pretreated embryonic suspension cells in the Agrobacterium tumefaciens culture medium, shake gently and let it stand for about 5 minutes, aspirate the Agrobacterium culture medium with a pipette, and then use a surgical blade to spread the cell mass on the filter paper surface on the MS solid plate medium containing 30 mg / L AS (acetosyringone) and 2.0 mg / L 2,4-D. Spread the cell mass as evenly as possible on the filter paper so that it is in full contact with the culture medium, and seal the culture dish with a sealing film. Let it stand and culture for 3 days in the dark at 28°C. Figure 1 Middle C).

[0097] (5) Use 20-mesh and 30-mesh sieves to select embryonic suspension cells suitable for transformation experiments: Select embryonic suspension cells that can pass through the 20-mesh sieve but not the 30-mesh sieve as transformation materials. Immerse a flask containing approximately 300 cell clusters in an ultrasonic cleaner (50W, 42KHz) for approximately 1 minute, then wash once with liquid 2,4-D medium and culture on a shaker for 3 days.

[0098] (6) After co-culture, transfer the embryonic cell mass into MS liquid medium containing 2 mg / L 2,4-D and 300 mg / L cefotaxime sodium (CS) and wash three times to remove any Agrobacterium tumefaciens adsorbed on the cell mass surface during infection. Then, transfer the mass into approximately 40 mL of MS liquid medium containing 2 mg / L 2,4-D and 300 mg / L CS and continue incubation for 1 week. If the medium becomes turbid around 3 days in between, replace the medium once.

[0099] (7) After delayed culture, the culture medium in the triangular flask was aspirated as clean as possible, and the cell clusters were gently transferred and spread on the filter paper on the MS solid culture medium containing 2 mg / L 2,4-D, 300 mg / L CS and the antibiotics of the vector selection marker. They were evenly spread (density was about 50 embryonic suspension cells per plate) so that each cell cluster could fully contact the culture medium. The selection culture was carried out in the dark at 28°C. After culturing for 2 weeks, the callus tissue with good growth, bright yellow color and hard texture was transferred to the surface of a single layer of filter paper on the MS solid culture medium containing 2 mg / L 2,4-D, 300 mg / L CS and the antibiotics of the vector selection marker to continue the selection culture. After that, the selection culture medium was changed every 2 weeks.

[0100] (8) After culturing embryonic suspension cells on the selective medium for 8 weeks, select embryonic cells with good growth status and transfer them to MS medium containing 1.0 mg / L ABA for cultivation. The light and temperature conditions in the culture room are constant: 28°C, 3000 Lx, 13 hours / day. Green mature somatic embryos can be obtained after 3-4 weeks of cultivation ( Figure 1 Middle D).

[0101] (9) After 2-4 weeks of induction, the mature somatic embryos that turned green on the ABA medium were transferred to MS solid medium and cultured at 27±1°C, 13 hours of light per day, and a light intensity of 3000 Lx for 4-8 weeks until complete transgenic plants were regenerated. The transgenic plants were subcultured on solid MS medium under a sterile environment ( Figure 1 E) and numbered sequentially. Transgenic plants were propagated every 6-8 weeks. Several roots, stems, and leaves of each plant were placed in a 1.5 mL centrifuge tube for GUS activity detection and genomic DNA extraction.

[0102] (7) The genomic DNA of the leaves of the proposed transgenic plants was extracted by the CTAB method. The extracted genomic DNA was used as a template, the genomic DNA of the wild-type plant was used as a negative control, and the plasmid pCAMBIA1300-IbJAZ10 was used as a positive control. PCR amplification was performed using IbJAZ10-FF-BglII and IbJAZ10-RR-PmlI as primers to obtain a PCR amplification product. If the PCR amplification product contained a band of about 570 bp, the corresponding sweet potato proposed transgenic plant was a sweet potato transgenic positive plant.

[0103] The results of electrophoresis amplification were shown in Figure 1 HJ, lane M is Maker, lane W is negative control water; lane P is positive control (recombinant plasmid pCAMBIA1300-IbJAZ10); lane WT is wild-type plant; lanes OE-J1 to OE-J27 are transgenic sweet potato plants transformed with pCAMBIA1300-IbJAZ10, Figure 1 As shown in Figures H and J, the target band of about 570 bp was amplified from lanes OE-J1 to OE-J27 and the positive control, indicating that the IbJAZ10 gene has been integrated into the sweet potato genome and proving that these regenerated plants are transgenic plants. Figure 1 As can be seen in Figures I and J, the expression of the IbJAZ10 gene was significantly reduced in the interfered plants, indicating that the present invention successfully interfered with the IbJAZ10 gene.

[0104] The transgenic sweet potato plants were propagated and transplanted into the field. The leaf phenotypes were observed. Figure 1 Middle F, harvested tubers Figure 1 In Figure G, it can be seen that the leaves of the overexpressing plants are larger than those of the disturbed plants. Although the tubers of the overexpressing plants have not undergone significant phenotype changes, they are more resistant to the disease after inoculation with soft rot pathogens than the wild type. Although the tubers of the disturbed plants have become thinner, their resistance to soft rot is greatly improved.

[0105] 4. Inoculation and identification of soft rot in transgenic plants

[0106] (1) Preparation of bacterial cells: 3 dishes of soft rot bacteria cultured on PDB medium for 3 days were scraped off the surface hyphae with an inoculating needle and placed in a conical flask containing 20 mL of PDB medium. A 1 cm diameter PDB solid medium disc was then added to the conical flask and shaken at 60 rpm for 2 h to allow a certain amount of bacterial cells to be stained on the disc for later use.

[0107] (2) Tuber preparation: Wash the tubers of wild-type plants, overexpression plants, and interference plants, dry them, cut them into slices about 1 cm thick, place them on a sterilized plate, and spray with sterile water to keep them moist;

[0108] (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 16 h, spraying with sterile water once during the inoculation period.

[0109] (4) Disease evaluation: Measure and count the disease diameter of potato chips 16 hours after inoculation.

[0110] The results are as follows Figure 2 As shown, the disease diameter of the overexpression plants was significantly higher than that of the wild-type plants, while the disease diameter of the interference plants was significantly lower than that of the overexpression plants, indicating that interfering with the IbJAZ10 gene in sweet potato can improve its resistance to soft rot.

[0111] Experiments have shown that interfering with the IbJAZ10 gene in sweet potato can improve its resistance to soft rot. Therefore, the sweet potato soft rot-related protein IbJAZ10 and its encoding gene have important theoretical significance in regulating plant soft rot resistance.

[0112] 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. Use of IbJAZ10 gene interference in any of the following: (1) Application in improving sweet potato soft rot resistance; (2) Application in the preparation of products that improve sweet potato soft rot resistance; (3) Application in breeding sweet potato varieties resistant to soft rot; (4) Application in the cultivation of sweet potatoes resistant to soft rot; The nucleotide sequence of the IbJAZ10 gene is shown in SEQ ID NO.

2.

2. A method for improving sweet potato soft rot resistance, characterized in that: Improving sweet potato soft rot resistance by interfering with the IbJAZ10 gene in sweet potato; The nucleotide sequence of the IbJAZ10 gene is shown in SEQ ID NO.

2.

3. A method for cultivating sweet potatoes resistant to soft rot, characterized in that: The method comprises the steps of interfering with the IbJAZ10 gene in the sweet potato, reducing the expression level of the IbJAZ10 gene, and obtaining the soft rot-resistant sweet potato; The nucleotide sequence of the IbJAZ10 gene is shown in SEQ ID NO.2.

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