Application of Protein IbNFYA3 in Regulating Resistance to Sweet Potato Soft Rot

Abstract

The present invention relates to the field of biotechnology, in particular to the application of the protein IbNFYA3 in regulating the resistance of sweet potato to soft rot. The present invention discovers for the first time that overexpressing the NFYA gene in plants can significantly increase the content of jasmonic acid and aspartic protease, thereby being able to effectively resist soft rot pathogens. Specifically, the present invention provides an IbNFYA3 gene encoding an NFYA transcription factor (protein IbNFYA3). After introducing this gene into sweet potato, transgenic sweet potato plants overexpressing the IbNFYA3 gene are obtained. When the transgenic sweet potato tubers are identified for their resistance to soft rot, it is found that compared with wild-type sweet potato, the transgenic sweet potato tubers have significantly enhanced resistance to soft rot. Thus, it can be seen that the IbNFYA3 protein and its encoding gene of the present invention can regulate the resistance of plants to soft rot and can be used to cultivate transgenic plants with high disease resistance.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of protein IbNFYA3 in regulating the resistance of sweet potato to soft rot disease. Background Art

[0002] Sweet potato has a high yield, strong environmental adaptability, and contains a large amount of starch. It can be used not only as an important food but also as feed, industrial raw materials, and new energy. Sweet potato is an excellent source of nutrients, including vitamins, potassium, iron, calcium, and minerals, and has medicinal value due to its anti-cancer, anti-diabetic, and anti-inflammatory activities, and is deeply loved by people.

[0003] Soft rot disease is a common disease of sweet potato during storage, mainly caused by Rhizopus stolonifer. The fungus invades from the wound and root pores of the sweet potato tuber, and secretes a variety of degrading enzymes into the plant tissue, quickly decomposing the middle lamella and other components in the sweet potato tuber cells, resulting in the collapse and rot of the sweet potato tissue, and spreading rapidly, often causing the whole cellar of sweet potato to rot, resulting in serious economic losses. Therefore, breeding disease-resistant sweet potato varieties has become one of the important measures to solve this disease.

[0004] In nature, plants have evolved a self-protection system to resist various biotic and abiotic stresses. Among them, NFYA transcription factors play an important role in the growth and development and disease resistance signaling system. Research shows that NFYA transcription factors regulate the antiviral ability of rice by interacting with plant hormone signaling pathways, especially by interacting with the important transcription factors OsMYC2 / 3 of the jasmonic acid (JA) pathway to regulate the JA pathway. NFYA transcription factors may also respond to abiotic stresses such as drought and high salt through the miR169 / NFYA module, and this module participates in the process of plants resisting abiotic stresses through the abscisic acid-mediated hormone signaling pathway. However, the relationship between the NFYA gene and soft rot fungi has not been reported. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of protein IbNFYA3 in regulating the resistance of sweet potato to soft rot disease to solve the problems existing in the above-mentioned prior art. Increasing the activity of the IbNFYA3 protein or increasing the expression level of its coding gene can improve the resistance of plants to soft rot disease.

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

[0007] The present invention provides an IbNFYA3 protein, and the amino acid sequence of the IbNFYA3 protein is as shown in SEQ ID NO.1. The SEQ ID NO.1 of the present invention is composed of 329 amino acid residues.

[0008] As an additional embodiment, the amino acid sequence of the IbNFYA3 protein of the present invention may be a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1; it may also be a protein derived from SEQ ID NO.1, which has undergone substitution and / or deletion and / or addition of one or several amino acid residues and is related to plant soft rot disease.

[0009] The present invention provides an IbNFYA3 gene encoding the above-mentioned IbNFYA3 protein, and the nucleotide sequence of the IbNFYA3 gene is as shown in SEQ ID NO.2. The SEQ ID NO.2 of the present invention consists of 990 nucleotides.

[0010] As an additional embodiment, the nucleotide sequence of the IbNFYA3 gene of the present invention may be a nucleic acid molecule whose coding region is SEQ ID NO.2; it may be a DNA molecule that hybridizes with SEQ ID NO.2 under stringent conditions or whose coding region is SEQ ID NO.2 and encodes the protein IbNFYA3; it may also be a DNA molecule that has 75% or more homology with SEQ ID NO.2 or whose coding region is SEQ ID NO.2 and encodes the IbNFYA3 protein. Among them, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; it may also be RNA, such as mRNA or hnRNA, etc.

[0011] In the present invention, those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence of the protein IbNFYA3 encoded by the present invention. Those artificially modified nucleotides with 75% or higher homology to the nucleotide sequence of the protein IbNFYA3 isolated from the present invention, as long as they encode the protein IbNFYA3 and are related to plant soft rot disease, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0012] The term "homology" used here refers to the sequence similarity with the natural nucleic acid sequence. "Homology" includes nucleotide sequences having 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher homology with the nucleotide sequence shown in SEQ ID NO.2 of the present invention. Homology can be evaluated by the naked eye or computer software. Using computer software, the homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences.

[0013] The present invention provides a biological material, and the biological material includes the above-mentioned IbNFYA3 gene.

[0014] Preferably, the biomaterial includes a recombinant expression vector and a recombinant microorganism.

[0015] As an additional aspect, the biomaterial of the present invention further includes an expression cassette and a transgenic plant cell line.

[0016] More preferably, the expression cassette includes a promoter, a nucleic acid molecule encoding the protein IbNFYA3, and a terminator; the promoter can be a CaMV35S promoter, a NOS promoter, or an OCS promoter; the terminator can be a NOS terminator or an OCS polyA terminator.

[0017] More preferably, the recombinant expression vector can be the recombinant expression vector pCAMBIA1300-IbNFYA3-GFP or the recombinant expression vector pCAMBIA1300-IbNFYA3;

[0018] More preferably, the recombinant expression vector pCAMBIA1300-IbNFYA3-GFP is obtained by the following steps:

[0019] The recombinant expression vector pCAMBIA1300-IbNFYA3-GFP obtained by inserting the IbNFYA3 gene between the multiple cloning sites of the vector pCAMBIA1300;

[0020] More preferably, the pCAMBIA1300 vector is digested with HindIII and EcoRI, the large fragment of the vector is recovered, the target gene sequence is amplified with the forward and reverse primers of the IbNFYA3 gene with the restriction endonucleases HindIII and EcoRI, and the recovered large fragment of the vector is ligated with the fragment containing the IbNFYA3 gene to obtain the recombinant expression vector pCAMBIA1300-IbNFYA3-GFP;

[0021] In the present invention, the recombinant microorganism can be obtained by introducing the recombinant expression vector into a starting microorganism; the recombinant microorganism can specifically be EHA105 / pCAMBIA1300-IbNFYA3; the EHA105 / pCAMBIA1300-IbNFYA3 is a recombinant agrobacterium obtained by transforming the recombinant plasmid pCAMBIA1300-IbNFYA3 into Agrobacterium tumefaciens EHA105.

[0022] In the present invention, the transgenic plant cell lines do not include propagation materials; the transgenic plants are understood to include not only the first-generation transgenic plants obtained by transforming the receptor plants with the IbNFYA3 gene (i.e., the coding gene of the IbNFYA3 protein), but also their progeny; for transgenic plants, the gene can be propagated in this species, or transferred into other varieties of the same species by conventional breeding techniques, especially including commercial varieties. The transgenic plants include seeds, callus, whole plants and cells.

[0023] As an additional aspect, the present invention provides the use of the above recombinant expression vector, the above recombinant microorganism, the above expression cassette or the above transgenic plant cell line in regulating the resistance of plants to soft rot.

[0024] More preferably, the regulation of the resistance of plants to soft rot can be to increase the resistance of plants to soft rot or to decrease the resistance of plants to soft rot.

[0025] More preferably, the disease against which the disease resistance is directed is soft rot;

[0026] and / or, the plant is sweet potato.

[0027] The present invention provides the use of the above IbNFYA3 protein, the above IbNFYA3 gene or the above biological material in increasing the disease resistance of plants.

[0028] Preferably, the disease against which the disease resistance is directed is soft rot;

[0029] and / or, the plant is sweet potato.

[0030] The present invention provides a method for increasing the disease resistance of plants, including the step of overexpressing the IbNFYA3 gene in the plants; the nucleotide sequence of the IbNFYA3 gene is as shown in SEQ ID NO.2.

[0031] Preferably, the disease against which the disease resistance is directed is soft rot;

[0032] and / or, the plant is sweet potato.

[0033] As an additional aspect, the present invention provides the use of the above recombinant expression vector, the above recombinant microorganism, the above expression cassette or the above transgenic plant cell line in cultivating transgenic plants with altered resistance to soft rot.

[0034] More preferably, the alteration of the resistance to soft rot can be an increase in the resistance to soft rot or a decrease in the resistance to soft rot.

[0035] As an additional solution, the method for transgenic plants with altered soft rot resistance in the present invention specifically includes the step of introducing a substance that enhances the expression and / or activity of the protein IbNFYA3 into a recipient plant to obtain a transgenic plant; the transgenic plant has enhanced soft rot resistance compared to the recipient plant.

[0036] In the above method for cultivating transgenic plants, the "introducing a substance that enhances the expression and / or activity of the protein IbNFYA3 into a recipient plant" can be achieved by introducing a nucleic acid molecule encoding the protein IbNFYA3 into the recipient plant.

[0037] In the above method for cultivating transgenic plants, the "introducing a nucleic acid molecule encoding the protein IbNFYA3 into a recipient plant" can be achieved by introducing a recombinant expression vector into the recipient plant; the recombinant expression vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein IbNFYA3 into an expression vector or a cloning vector; specifically, the recombinant expression vector can be the recombinant plasmid pCB-IbNFYA3.

[0038] Further preferably, the disease against which the disease resistance is targeted is soft rot;

[0039] and / or, the plant is sweet potato.

[0040] The present invention provides the application of the above IbNFYA3 protein, the above IbNFYA3 gene, or the above biological material in cultivating transgenic plants with high disease resistance.

[0041] Further preferably, the disease against which the disease resistance is targeted is soft rot;

[0042] and / or, the plant is sweet potato.

[0043] The present invention provides a method for cultivating transgenic plants with high disease resistance, including the step of overexpressing the IbNFYA3 gene in the plant to obtain the transgenic plant with high disease resistance; the nucleotide sequence of the IbNFYA3 gene is as shown in SEQ ID NO.2.

[0044] Further preferably, the disease against which the disease resistance is targeted is soft rot;

[0045] and / or, the plant is sweet potato.

[0046] The present invention discloses the following technical effects:

[0047] The present invention discovers for the first time that overexpressing the NFYA gene in plants can significantly increase the content of jasmonic acid and aspartic protease, thereby being able to effectively resist soft rot pathogens. Specifically, the present invention provides an IbNFYA3 gene encoding an NFYA transcription factor (protein IbNFYA3). After introducing this gene into sweet potato, transgenic sweet potato plants overexpressing the IbNFYA3 gene are obtained. When the transgenic potato tubers are identified for soft rot resistance, it is found that compared with the wild type, the transgenic potato tubers have significantly enhanced resistance to soft rot. Thus, it can be seen that the IbNFYA3 protein and its encoding gene of the present invention can regulate the resistance of plants to soft rot and can be used to cultivate transgenic plants with high disease resistance. In short, the IbNFYA3 protein and its encoding gene provided by the present invention have important theoretical significance and application value in the research on the soft rot resistance of sweet potato. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0049] Figure 1 PCR detection of transgenic plants; wherein, A is callus cultured in MS solid medium containing 2 mg / L 2,4-D; B is embryogenic suspension cells cultured in MS liquid medium containing 2 mg / L 2,4-D; C is embryogenic callus with Hyg resistance cultured in MS solid plate containing 100 mg / L CS, 11 mg / L Hyg and 2 mg / L 2,4-D; D is putative transgenic plants regenerated in MS solid medium containing 1 mg / L ABA and 100 mg / L CS; E is transgenic IbNFYA3 sweet potato plants subcultured on MS solid medium; F is the phenotypic situation of transgenic sweet potato leaves overexpressing the IbNFYA3 gene; G is the phenotypic situation of transgenic sweet potato tubers overexpressing the IbNFYA3 gene; H is the PCR detection results of different plants, lane M is Maker, lane W is negative control water, lane P is positive control (recombinant plasmid pCAMBIA1300-IbNFYA3), lane WT is wild type sweet potato plants, and the remaining lanes are transgenic sweet potato plants overexpressing the IbNFYA3 gene transformed with pCAMBIA1300-IbNFYA3; I is the expression level of the IbNFYA3 gene in different plants;

[0050] Figure 2 Soft rot resistance identification of transgenic IbNFYA3 sweet potato tubers; wherein, A is the picture of the disease after inoculating soft rot pathogens on slices; B is the statistical chart of the disease diameter. Detailed Embodiments

[0051] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0052] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0054] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0055] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0056] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0057] The materials, reagents, etc. used in the following examples can all be obtained commercially unless otherwise specified.

[0058] Example 1 Obtaining and Function Verification of Proteins Related to Sweet Potato Soft Rot and Their Encoding Genes

[0059] I. Obtaining of Proteins Related to Sweet Potato Soft Rot and Their Encoding Genes

[0060] 1. Total RNA extraction from sweet potato: Take 1 g of young leaves of the sweet potato line Nongdabai (NDB), grind them into powder in liquid nitrogen, add them to a 2 mL centrifuge tube, extract the total RNA from sweet potato using the Trizon method, and reverse transcribe the first-strand cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser kit.

[0061] 2. With the help of the sweet potato stress resistance gene IbJAZ10 that has been studied in the laboratory, the interacting gene IbNFYA3 was screened through the sweet potato yeast two-hybrid library, and it was aligned in the Sweetpotato Garden library to obtain the EST sequence shown in SEQ ID NO.2. According to the nucleotide sequence of the EST sequence, primers IbNFYA3-F and IbNFYA3-R were designed and artificially synthesized, and the sequences are as follows:

[0062] IbNFYA3-F: 5’-ATGCTAAATTTCTCATTCTCCAAG-3’, SEQ ID NO.3;

[0063] IbNFYA3-R: 5’-TCAGGTTCTTCTACGCACAAA-3’, SEQ ID NO.4;

[0064] 3. Using the cDNA obtained in step 1 as a template and the IbNFYA3-F and IbNFYA3-R synthesized in step 2 as primers, PCR amplification was carried out to obtain a PCR amplification fragment product of about 990 bp and sequenced.

[0065] The results showed that the nucleotide sequence of the PCR amplification product obtained in step 3 is as shown in SEQ ID NO.2, specifically: ATGCTAAATTTCTCATTCTCCAAGAAAGAAGGTGATCAGACCGCTGCTCAGTCATTCACACCTATGTCTATAACTAGCTCATCGCTGTGGAATTCCACTGATCAACCAGAAAATCCTCTTTCTGAGAGTGCAGATACTGGACAAAAATCTGCACCCCAACGTGGTTTCTGCATGAAGCAAACAGAATCTCAACTTCAAGATCAGGATAACACTTCAACTCTATCGACTGACCAATCTCATCAAACAGTGGCTGCAATGACACCAAGCAATTGTCACAAAGTTGGACCTCAACCTGGCTATGCTGAAATCCATGAAAGGCAAATGAAGGATTGCTCTATAAAGCCATCTCAGCCACATTTAGATGAGGATTGCACCATTCATCAAGGACAATTGGATTTTAGCCAGTCCATGGCTTGCCTATCTTGGACTGAACCATATCTTGGGAGGCTGGTAGCTACTTATGGACCAAATGGTATTATTTATCCTCAAATGGTTGGAATTGTACCTGCAAGAATGCCACTTCCTTCTGAATGCGCAGAAAGCATACCTATTTATGTTAATGCAAAACAATATCGCGCAATTCTCAGACGGCGAGAAATCCGTGCTAAGCTCGAGGCTGAGAACAAAGTTGTCAAAGTGAGGAAGCCATATCTTCACGAGTCTCGACATGCTCATGCCTTGAAGAGAGCAAGGGGTTCTGGAGGCCGTTTTCTGAACAAAACCGAACTACAGCAACTCAAATCTGCTGCATCTCCGACCCATGGGAAAAATATCTCTAACCAAAAGGGCGGTGGAGACATATCTGGCTCTCAACTCCAGCACTCAGAAAGCGGTAGTTGGGGGACTTCTACCCCTTCCGGCTCTGATGTCACAAGCATCTTTAGTGGTGATGGCATTTTCCAGCAGCCGGAGTTTAGAGTCTCCAGCTCTCCTTATCACATGGGCGTTTCTATGCACGAGGCTGAAAACTTTGTGCGTAGAAGAACC TGA , the gene shown by this sequence was named as IbNFYA3 gene, and the protein encoded by it was named as IbNFYA3 protein or protein IbNFYA3. The amino acid sequence is as shown in SEQ ID NO.1, specifically: MLNFSFSKKEGDQTAAQSFTPMSITSSSLWNSTDQPENPLSESADTGQKSAPQRGFCMKQTESQLQDQDNTSTLSTDQSHQTVAAMTPSNCHKVGPQPGYAEIHERQMKDCSIKPSQPHLDEDCTIHQGQLDFSQSMACLSWTEPYLGRLVATYGPNGIIYPQMVGIVPARMPLPSECAESIPIYVNAKQYRAILRRREIRAKLEAENKVVKVRKPYLHESRHAHALKRARGSGGRFLNKTELQQLKSAASPTHGKNISNQKGGGDISGSQLQHSESGSWGTSTPSGSDVTSIFSGDGIFQQPEFRVSSSPYHMGVSMHEAENFVRRRT。

[0066] II. Application of IbNFYA3 protein in regulating the resistance of sweet potato to soft rot

[0067] 1. Construction of plant expression vector

[0068] According to the nucleotide sequence of sweet potato IbNFYA3 gene (SEQ ID NO.2), primer sequences for amplifying the complete coding sequence were designed. Restriction enzyme cleavage sites of BglII and PmlI were introduced into the forward and reverse primers respectively. The primer sequences are as follows:

[0069] IbNFYA3-FF-BglII: 5’-ACTCTTGACCATGGT AGATCT ATGCTAAATTTCTCATTCTCCAA G-3’ (the underlined part is the BglII restriction enzyme cleavage site), SEQ ID NO.5;

[0070] IbNFYA3-RR-PmlI: 5’-GTCACCTGTAATTCA CACGTG TCAGGTTCTTCTACGCACAAA-3’ (the underlined part is the PmlI restriction enzyme cleavage site), SEQ ID NO.6;

[0071] Using the artificially synthesized SEQ ID NO.2 as a template, after PCR amplification, the product was ligated to the pMD19-T vector, named pMD-IbNFYA3 vector, and sequenced with M13-F / R to ensure the correct reading frame of the nucleotides of the sweet potato IbNFYA3 gene and the restriction enzyme sites.

[0072] The pCAMBIA1300 vector was digested with HindIII and EcoRI double enzymes, and the large fragment of the vector was recovered. The pBI121 vector was digested with HindIII and EcoRI double enzymes, and the fragment containing the gusA gene was recovered. The recovered large fragment of the vector was ligated with the fragment containing the gusA gene to obtain the recombinant expression vector pCBGUS.

[0073] The vector pCBGUS was digested with BglII and PmlI, and the large fragment of the vector was recovered. At the same time, a fragment was amplified from the vector pMD-IbNFYA3 using the primers IbNFYA3-FF-BglII and IbNFYA3-RR-PmlI, and a fragment of about 990 bp was recovered. The recovered large fragment of the vector was ligated with the fragment of about 990 bp to obtain the target plasmid. The target plasmid was transformed into Escherichia coli DH5α and cultured at 37 °C for 20 h. PCR analysis and restriction enzyme identification of the recombinant expression vector were carried out, and sequencing verification was also carried out. The sequencing results showed that the sequence shown in SEQ ID NO.2 was inserted between the BglII and PmlI restriction enzyme sites of the recombinant expression vector pCBGUS, indicating that the recombinant expression vector was correctly constructed, and the recombinant plasmid pCAMBIA1300-IbNFYA3 expressed the protein IbNFYA3 shown in SEQ ID NO.1.

[0074] The recombinant plasmid pCAMBIA1300-IbNFYA3 has an expression cassette, and the nucleotide sequence of this expression cassette contains the CaMV35S promoter, the coding gene of the IbNFYA3 protein, and the NOS terminator.

[0075] 2. Transformation of the plant expression vector into Agrobacterium

[0076] The process is as shown in A-G in Figure 1 and is specifically as follows:

[0077] (1) The prepared competent cells of Agrobacterium EHA105 were thawed on ice, 2 μg of the extracted pCAMBIA1300-IbNFYA3 plasmid was added, the tube wall was flicked gently to mix evenly, and incubated on ice for 10 min;

[0078] (2) Quick-frozen in liquid nitrogen for 5 min, water-bathed at 37 °C for 10 min, and incubated on ice for 5 min;

[0079] (3) 600 μL of liquid LB medium was added, and cultured at 28 °C and 200 rpm for 5 h;

[0080] (4) Spread 200 μL of the bacterial solution onto an LB solid medium containing 100 μg / mL ampicillin and 100 μg / mL rifampicin.

[0081] (5) Incubate in the dark at 28 °C for 2 days in an inverted position. Take an appropriate amount of Agrobacterium and culture it in liquid LB medium for later use. That is, obtain the Agrobacterium solution transformed with the pCAMBIA1300-IbNFYA3 vector, and name the recombinant Agrobacterium EHA105 / pCAMBIA1300-IbNFYA3.

[0082] (6) Genetic transformation and regeneration of sweet potato

[0083] Use the Agrobacterium-mediated method to introduce EHA105 / pCAMBIA1300-IbNFYA3 into the sweet potato Lizixiang. The specific method is as follows:

[0084] 6.1) Gently rinse the shoot tips retrieved from the greenhouse with distilled water to remove dust, residual leaves, etc. on the surface of the shoot tips; remove the larger leaves on the explants, leaving the shoot segments with shoot tips, with a length of about 1 - 1.5 cm being appropriate; first soak and disinfect in 70% ethanol solution for 30 s, then quickly transfer the shoot tips into 2% sodium hypochlorite solution for disinfection for 5 min, gently shake during this period to ensure full contact between the shoot tips and the sodium hypochlorite solution; after disinfection is completed, pour out the waste liquid and rinse with sterile water 3 times to remove the ethanol and sodium hypochlorite solution that may remain on the shoot tips.

[0085] 6.2) Immerse the disinfected shoot segments with shoot tips in sterile water for the dissection of shoot apical meristems; use forceps to pick out the shoot tips immersed in sterile water and place them on a culture dish. Under a binocular stereomicroscope (20×), cut off the leaves and leaf primordia surrounding the shoot apical meristem from the outside to the inside (1 - 2 leaf primordia closely attached to the meristem can be retained), carefully cut off the exposed meristem (about 0.5 mm) and inoculate it into a 100 mL Erlenmeyer flask containing MS solid medium with 2 mg / L 2,4-D, and culture in the dark to induce embryogenic callus ( Figure 1 in A).

[0086] 6.3) At room temperature of 27 ± 1 °C, after 6 - 8 weeks of dark culture, embryogenic callus will be successively formed from the shoot apical meristems. Gently transfer the well-developed embryogenic callus to MS liquid medium containing 2 mg / L 2,4-D, and use forceps to break it into small cell clusters, and culture at 100 rpm, 27 ± 1 °C, and 500 Lx light (13 h per day). After 8 - 12 weeks of culture, select the suspension cells with good growth status, rapid propagation, and bright yellow color as the recipients for sweet potato genetic transformation, and carry out subsequent transformation work ( Figure 1 in B).

[0087] 6.4) Immerse the pre-treated embryogenic suspension cells in the Agrobacterium tumefaciens solution, gently shake well and then let stand for about 5 min. Suck out the Agrobacterium tumefaciens solution with a pipette, and then use a surgical blade to spread the cell clusters flat on the filter paper surface of the MS solid plate medium containing 100 mg / L CS, 11 mg / L Hyg and 2 mg / L 2,4-D. Try to disperse and evenly spread the cell clusters on the filter paper so that they can fully contact the medium, and seal the culture dish with a sealing film. Incubate at 28 °C in the dark for 3 d ( Figure 1 in C).

[0088] 6.5) Use 20-mesh and 30-mesh sieves to select sweet potato embryogenic suspension cells suitable for transformation experiments: Select embryogenic suspension cells that can pass through the 20-mesh sieve and cannot pass through the 30-mesh sieve as transformation materials. Immerse the Erlenmeyer flask containing about 300 cell clusters in an ultrasonic cleaner (50 w, 42 KHz) and ultrasonically treat for about 1 min, then wash once with liquid 2,4-D medium and culture on a shaker for 3 d.

[0089] 6.6) Transfer the co-cultured embryogenic cell clusters into MS liquid medium containing 1 mg / L ABA and 100 mg / L cefotaxime sodium (CS) and wash 3 times to try to wash off the Agrobacterium tumefaciens adsorbed on the surface of the cell clusters during infection. Then transfer them into about 40 mL of MS liquid medium containing 1 mg / L ABA and 100 mg / L CS for delayed culture for 1 w. If the medium becomes turbid about 3 d in the middle, the medium can be replaced once.

[0090] 6.7) Try to suck out the medium in the Erlenmeyer flask after delayed culture as clean as possible, gently transfer and spread the cell clusters on the filter paper on the MS solid medium containing 1 mg / L ABA, 100 mg / L CS and the antibiotic of the vector selection marker, and spread them evenly (the density is about 50 embryogenic suspension cells per plate) so that each cell cluster can fully contact the medium, and carry out selection culture in the dark at 28 °C. After culturing for 2 w, transfer the well-grown callus that is bright yellow and relatively hard to the single-layer filter paper surface of the MS solid medium containing 1 mg / L ABA, 100 mg / L CS and the antibiotic of the vector selection marker to continue selection culture, and then replace the selection medium once every 2 w.

[0091] 6.8) After culturing the embryogenic suspension cells on the selection medium for 8 w, select the well-grown embryogenic cells and transfer them to MS liquid medium containing 1 mg / L ABA and 100 mg / L CS for culture. The light and temperature conditions in the culture room are constant: 28 °C, 3000 Lx, 13 h / d. Green mature somatic embryos can be obtained after culturing for 3 - 4 w.

[0092] 6.9) Transfer the mature somatic embryos that turn green on the ABA medium after 2 - 4 weeks of induction to the MS solid medium containing 1 mg / L ABA and 100 mg / L CS, and culture them at 27 ± 1 °C under a 13 - hour light per day with a light intensity of 3000 Lx for 4 - 8 weeks until complete transgenic plants are regenerated ( Figure 1 D in Figure 1 ). Sub - culture the transgenic plants on the MS solid medium in a sterile environment, number them in sequence, and multiply the transgenic plants once every 6 - 8 weeks ( Figure 1 E in

[0093] ) to obtain transgenic sweet potatoes overexpressing the IbNFYA3 gene. The leaf phenotypes and tuber phenotypes of the transgenic sweet potatoes overexpressing the IbNFYA3 gene are shown in

[0094] F and G in Figure 1 . At the same time, place several roots, stems, and leaves of each plant in 1.5 - mL centrifuge tubes for GUS activity detection and genomic DNA extraction. Figure 1 (7) Extract the genomic DNA of the leaves of transgenic plants overexpressing the IbNFYA3 gene by the CTAB method. Using the extracted genomic DNA as a template, water and wild - type plants as negative controls, and plasmid pCB - IbNFYA3 as a positive control, perform PCR amplification with IbNFYA3 - FF - BglII and IbNFYA3 - RR - PmlI as primers to obtain PCR amplification products; if the PCR amplification products contain a band of approximately 1000 bp, the corresponding transgenic sweet potato plants are transgenic positive plants, that is, transgenic sweet potatoes overexpressing the IbNFYA3 gene.

[0095] 3. Inoculation identification of soft rot disease of transgenic plants

[0096] The disease-free and healthy transgenic potato lines and wild-type potato tubers were washed and air-dried, and then inoculated with soft rot pathogens to identify their resistance to soft rot. The specific inoculation method is as follows:

[0097] (1) Preparation of bacterial cells: Three petri dishes containing PDB medium were used to culture soft rot pathogens for 3 days. Then, the surface mycelium was scraped off with an inoculation needle and placed into a triangular flask containing 20 mL of PDB medium. A PDB solid medium disc with a diameter of 1 cm was added to the triangular flask, and the flask was shaken on a shaker at 60 rpm for 2 h to allow a certain amount of bacterial cells to adhere to the disc for later use;

[0098] (2) Preparation of potato tubers: The potato tubers were washed clean, air-dried, and then sliced into potato chips with a thickness of about 1 cm, which were placed on a sterilized petri dish and sprayed with sterile water to keep them moist;

[0099] (3) Inoculation and identification: One disc was inoculated in the middle of the potato chip. After inoculation, the petri dish was placed in an incubator at 28 °C for 16 h, and sterile water was sprayed once during this period to keep it moist;

[0100] (4) Disease evaluation: The disease diameter of the potato chip was measured 16 h after inoculation. The results are as Figure 2 shown. The results showed that: The experiment proved that overexpression of the IbNFYA3 gene in sweet potato enhanced its resistance to soft rot.

[0101] In summary, the sweet potato soft rot-related protein IbNFYA3 and its encoding gene have important theoretical significance in regulating plant resistance to soft rot.

[0102] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An IbNFYA3 protein, characterized in that, The amino acid sequence of the IbNFYA3 protein is shown in SEQ ID NO.

1.

2. The IbNFYA3 gene encoding the IbNFYA3 protein according to claim 1, characterized in that, The nucleotide sequence of the IbNFYA3 gene is shown in SEQ ID NO.

2.

3. A biological material, characterized in that, The biological material includes the IbNFYA3 gene described in claim 2; the biological material is a recombinant expression vector and a recombinant microorganism.

4. Use of the IbNFYA3 protein according to claim 1, the IbNFYA3 gene according to claim 2, or the biological material according to claim 3 in improving the disease resistance of plants, characterized in that, The disease against which the disease resistance is directed is soft rot; The plant is sweet potato.

5. A method for improving the disease resistance of plants, characterized in that, It includes the step of overexpressing the IbNFYA3 gene in the plant; the nucleotide sequence of the IbNFYA3 gene is shown in SEQ ID NO.2; The disease against which the disease resistance is directed is soft rot; The plant is sweet potato.

6. Use of the IbNFYA3 protein according to claim 1, the IbNFYA3 gene according to claim 2, or the biological material according to claim 3 in cultivating transgenic plants with high disease resistance, characterized in that, The disease against which the disease resistance is directed is soft rot; The plant is sweet potato.

7. A method for cultivating a transgenic plant with high disease resistance, characterized in that, It includes the step of overexpressing the IbNFYA3 gene in the plant to obtain the transgenic plant with high disease resistance; the nucleotide sequence of the IbNFYA3 gene is shown in SEQ ID NO.2; The disease against which the disease resistance is directed is soft rot; The plant is sweet potato.

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