Method for improving soft rot resistance of sweet potatoes

Abstract

The invention discloses a method for improving soft rot resistance of sweet potatoes, and belongs to the technical field of plant genetic engineering. The invention provides application of IbJAZ10 protein in any one of the following items: (1) application in regulation and control of sweet potato soft rot; (2) application in preparation of products for regulating and controlling sweet potato soft rot; (3) application in soft rot resistant sweet potato breeding; (4) application in cultivation of soft rot resistant sweet potatoes; the amino acid sequence of the IbJAZ10 protein is as shown in SEQ ID NO. 3. Experiments prove that compared with a wild type, the IbJAZ10 gene interference plant has the advantage that the disease resistance of the IbJAZ10 gene interference plant is obviously improved. The IbJAZ10 protein and the coding gene thereof provided by the invention have important theoretical significance and application value in the research on the soft rot resistance of sweet potatoes.

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 have high yields, strong ability to adapt to the environment, and contain a lot of starch. They can be used not only as an important food, but also as feed, industrial raw materials and new energy sources. Sweet potatoes are an excellent source of nutrients, including vitamins, potassium, iron, calcium and minerals. They have medicinal value due to their anti-cancer, anti-diabetic and anti-inflammatory activities and are deeply loved by people.

[0003] Soft rot is a common disease of sweet potatoes during storage, mainly caused by Rhizopus stolonifer, which invades from the wounds and root pores of the tubers and secretes a variety of decomposing enzymes into the plant tissues, which quickly decompose the gelatin layer and other components in the tuber cells, causing the sweet potato tissues to disintegrate and rot, and spread rapidly, often causing the whole cellar to rot, resulting in serious economic losses. Therefore, breeding new disease-resistant sweet potato varieties has become one of the important measures to solve 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 potatoes resistant to soft rot.

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

[0006] The present invention provides the use of 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 sweet potato breeding for resistance to soft rot;

[0010] (4) Application in breeding 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 items:

[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 sweet potato breeding for resistance to soft rot;

[0016] (4) Application in breeding 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 coding 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 sweet potato breeding for resistance to soft rot;

[0022] (4) Application in breeding 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 sweet potato breeding for resistance to soft rot;

[0028] (4) Application in breeding 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 regulating sweet potato soft rot, comprising any 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 potato to soft rot by overexpressing the IbJAZ10 gene in sweet potato;

[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 an 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, and 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 resistance of the transgenic plants to soft rot is significantly reduced; and compared with the wild type, the disease resistance of the plants interfered with the IbJAZ10 gene is significantly improved. The IbJAZ10 protein and the gene encoding the same 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0040] Figure 1 Obtaining and identifying transgenic plants; A: Callus cultured in MS solid medium containing 2 mg / L 2,4-D; B: Embryonic suspension cells cultured in MS liquid medium containing 2 mg / L 2,4-D; C: Embryonic callus cultured in MS solid medium containing 300 mg / LCS and 2 mg / L 2,4-D; D: Pseudo-transgenic plants regenerated in MS solid medium containing 1 mg / L ABA; E: Transgenic IbJAZ10 sweet potato plants subcultured on MS solid medium, the left picture is the overexpression plant, and the right picture is the interference plant; FG: Phenotypes of leaves (F) and sweet potato tubers (G) of overexpression and interference plants; HI: Gene electrophoresis results of overexpression plants 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 disease 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 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 into a 2 mL centrifuge tube. The total RNA of the sweet potato was extracted by 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] With the help of the sweet potato stress resistance gene IbBBX24 (reference patent CN109306000A-stress resistance-related protein IbBBX24 and its encoding gene and application) that has been studied in the laboratory, the interactive gene IbJAZ10 was screened by the sweet potato yeast two-hybrid library, and it was compared in the Sweetpotato Garden library to obtain the EST sequence (nucleotide sequence is shown in SEQ ID NO.1). According to the nucleotide sequence of the EST sequence, primers IbJAZ10-F and IbJAZ10-R were designed and artificially synthesized, and the sequences are as follows.

[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 about 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] According to the coding sequence of sweet potato IbJAZ10 protein nucleotide, the primer sequence for amplifying the complete coding sequence was designed, and the forward and reverse primers were introduced into the 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 part 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 connected to the pMD19-T vector, named pMD-IbJAZ10 vector, and M13-F / R sequencing was performed to ensure the correct reading frame and restriction site of the sweet potato IbJAZ10 protein nucleotide.

[0071] The pCAMBIA1300 vector was double-digested with HindIII and EcoRI to recover the large vector fragment, and 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. At the same time, the fragment was amplified from the vector pMD-IbJAZ10 using primers IbJAZ10-FF and IbJAZ10-RR, and a fragment of about 570 bp was recovered. The recovered large vector fragment was connected with the fragment of about 570 bp to obtain the target plasmid. The target plasmid was transformed into Escherichia coli DH5α, cultured at 37°C for 20 hours, and the recombinant vector was analyzed by PCR and digested, and sequenced for verification. The sequencing results showed that the sequence shown in SEQ ID NO.2 was inserted between the BglII and PmlI restriction sites of the vector pCBGUS, indicating that the recombinant vector was constructed correctly and 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 vector

[0075] According to the coding sequence of sweet potato IbJAZ10 protein nucleotide, the primer sequence for amplifying the interference fragment was designed. The primer sequence is 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] The plasmid pFGC-5941 was subjected to a double restriction digestion reaction of Xho I and Swa I, and was connected to the forward target sequence of the pFGC-5941 homology arm IbJAZ10 containing the double restriction sites of Xho I and Swa I, and transformed into the competent E. coli cell DH5α to obtain a recombinant cloning vector containing the forward fragment of IbJAZ10, which was named IbJAZ10-5941F after correct sequencing. Then, IbJAZ10-5941F was subjected to a double restriction digestion reaction of BamH I and Xba I, and the reverse target sequence of the pFGC5941 homology arm IbJAZ10 containing the double restriction sites of BamH I and Xba I was connected, and then transformed into E. coli, and finally a recombinant RNAi expression vector of the reverse repeat sequence was obtained, which was named pFGC-IbJAZ10-5941 after correct sequencing.

[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, flick the tube wall to mix, and place 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 culture at 28°C, 200 rpm for 5 h;

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

[0090] (5) Incubate the 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, thus obtaining the Agrobacterium culture solution introduced with the pCB-IbJAZ10 vector. The recombinant Agrobacterium was 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 the sweet potato chestnut fragrance by Agrobacterium-mediated method. The specific method is as follows:

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

[0094] (2) Soak the disinfected 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 in a 100 mL Erlenmeyer flask containing 2.0 mg / L 2,4-D MS solid medium and culture it in the dark to induce embryonic callus ( Figure 1 (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. Culture is carried out 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 are 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 to make it fully contact with the culture medium, and seal the culture dish with a sealing film. Let it stand and co-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 sweet potato embryonic suspension cells suitable for transformation experiments: Select embryonic suspension cells that can pass through a 20-mesh sieve but cannot pass through a 30-mesh sieve as transformation materials. Immerse a flask containing about 300 cell clusters in an ultrasonic cleaner (50w, 42KHz) for about 1 min, then wash once with liquid 2,4-D culture 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 it three times to remove the Agrobacterium tumefaciens adsorbed on the surface of the cell mass during infection. Then transfer it into about 40 mL of MS liquid medium containing 2 mg / L 2,4-D and 300 mg / L CS and culture it for 1 week. If the medium becomes turbid around 3 days in the middle, replace the medium once.

[0099] (7) After delayed culture, the culture medium in the flask was removed as cleanly as possible, and the cell clusters were gently transferred and spread evenly on the filter paper on the MS solid culture medium containing 2 mg / L 2,4-D, 300 mg / L CS and antibiotics for vector selection markers. The cells were evenly spread (density was about 50 embryonic suspension cells per plate) so that each cell cluster could fully contact the culture medium, and 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 antibiotics for vector selection markers for continued selection culture, and the selection medium was replaced every 2 weeks.

[0100] (8) After culturing embryonic suspension cells on the selective medium for 8 weeks, embryonic cells with good growth status were selected and transferred to MS medium containing 1.0 mg / L ABA for culture. The light and temperature conditions in the culture room were constant: 28°C, 3000 Lx, 13h / d. Green mature somatic embryos were obtained after 3-4 weeks of culture ( 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 for 4-8 weeks at 27±1°C, 13 hours of light per day, and a light intensity of 3000 Lx until complete transgenic plants were regenerated. The transgenic plants were subcultured on solid MS medium under a sterile environment ( Figure 1 E), and numbered them in sequence, and the intended transgenic plants were propagated once every 6-8 weeks. At the same time, 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, and the extracted genomic DNA was used as a template, the genomic DNA of the wild-type plants 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 contains a band of about 570 bp, the corresponding sweet potato proposed transgenic plant is a sweet potato transgenic positive plant.

[0103] The results of electrophoresis detection amplification are shown in Figure 1 In 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 in 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 identified were propagated and transplanted into the field. The leaf phenotypes in the field were observed. Figure 1 Middle F, harvested tubers Figure 1 In Figure G, it can be seen that the leaves of the overexpression plants are larger than those of the interference plants. Although the tubers of the overexpression 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 interference plants have become thinner, their resistance to soft rot has been greatly improved.

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

[0106] (1) Preparation of bacterial cells: Use an inoculation needle to scrape the surface hyphae of 3 dishes of soft rot bacteria cultured on PDB medium for 3 days and place them in a conical flask containing 20 mL of PDB medium. Then add a 1 cm diameter PDB solid medium disc to the conical flask and shake it 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 them 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 sterile water once to keep it moist;

[0109] (4) Disease evaluation: The disease diameter of the potato chips was measured and counted 16 hours after inoculation.

[0110] The results are as follows Figure 2 As shown, it can be seen that the disease diameter of the overexpression plant is significantly higher than that of the wild-type plant, while the disease diameter of the interference plant is significantly lower than that of the overexpression plant, 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of IbJAZ10 protein in any of the following: (1) Application in regulating sweet potato soft rot; (2) Application in the preparation of products for regulating sweet potato soft rot; (3) Application in sweet potato breeding for resistance to soft rot; (4) Application in breeding sweet potatoes resistant to soft rot; The amino acid sequence of the IbJAZ10 protein is shown in SEQ ID NO.

3.

2. Use of the gene IbJAZ10 encoding the IbJAZ10 protein in any of the following: (1) Application in regulating sweet potato soft rot; (2) Application in the preparation of products for regulating sweet potato soft rot; (3) Application in sweet potato breeding for resistance to soft rot; (4) Application in breeding sweet potatoes resistant to soft rot; The nucleotide sequence encoding the gene IbJAZ10 is shown in SEQ ID NO.

2.

3. Use of a recombinant vector containing the coding gene IbJAZ10 in any of the following: (1) Application in regulating sweet potato soft rot; (2) Application in the preparation of products for regulating sweet potato soft rot; (3) Application in sweet potato breeding for resistance to soft rot; (4) Application in breeding sweet potatoes resistant to soft rot; The nucleotide sequence encoding the gene IbJAZ10 is shown in SEQ ID NO.

2.

4. Use of host bacteria containing recombinant vectors in any of the following: (1) Application in regulating sweet potato soft rot; (2) Application in the preparation of products for regulating sweet potato soft rot; (3) Application in sweet potato breeding for resistance to soft rot; (4) Application in breeding sweet potatoes resistant to soft rot; 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.

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, characterized in that: This includes any of the following methods: (1) Improving the resistance of sweet potato to soft rot by interfering with the IbJAZ10 gene in sweet potato; (2) reducing the resistance of sweet potato to soft rot by overexpressing the IbJAZ10 gene in sweet potato; The nucleotide sequence of the IbJAZ10 gene is shown in SEQ ID NO.

2.

7. 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 sweet potato resistant to soft rot; The nucleotide sequence of the IbJAZ10 gene is shown in SEQ ID NO.2.

Images