Application of protein IbNFYA3 in regulation and control of soft rot resistance of sweet potatoes

Abstract

The invention relates to the technical field of biology, in particular to application of protein IbNFYA3 in regulation and control of soft rot resistance of sweet potatoes. It is found for the first time that the content of jasmonic acid and the content of aspartic protease can be remarkably increased through overexpression of the NFYA gene in a plant, so that soft rot bacteria can be effectively resisted, specifically, the invention provides an IbNFYA3 gene for coding an NFYA transcription factor (protein IbNFYA3), the gene is introduced into sweet potatoes to obtain a transgenic sweet potato plant with the overexpression of the IbNFYA3 gene, and the transgenic sweet potato plant has the advantages that the content of jasmonic acid and the content of aspartic protease can be effectively resisted; soft rot resistance identification is carried out on the transgenic sweet potato blocks, and it is found that compared with wild sweet potatoes, the soft rot resistance of the transgenic sweet potato blocks is obviously enhanced. Therefore, the IbNFYA3 protein and the coding gene thereof can regulate and control the soft rot resistance of plants, and can be used for cultivating transgenic plants with high disease resistance.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of protein IbNFYA3 in regulating sweet potato soft rot resistance. 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 of sweet potatoes to rot, causing 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 which NFYA transcription factors play an important role in growth and development and disease resistance signaling systems. Studies have shown that NFYA transcription factors regulate the antiviral ability of rice by interacting with plant hormone signaling pathways, especially interacting with OsMYC2 / 3, an important transcription factor in the jasmonic acid (JA) pathway. NFYA transcription factors may also respond to plant abiotic stresses, such as drought and high salt, through the miR169 / NFYA module. This module participates in the process of plant resistance to abiotic stresses through the abscisic acid-mediated hormone signaling pathway. However, the relationship between the NFYA gene and soft rot pathogens has not been reported. Summary of the invention

[0005] The purpose of the present invention is to provide an application of protein IbNFYA3 in regulating sweet potato soft rot resistance 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 encoding gene can improve the resistance of plants to soft rot.

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

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

[0008] As an additional scheme, the amino acid sequence of the IbNFYA3 protein described in the present invention can be a fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the amino acid sequence shown in SEQ ID NO.1; it can also be a protein derived from SEQ ID NO.1 in which one or several amino acid residues are substituted and / or deleted and / or added to the amino acid sequence shown in SEQ ID NO.1 and is related to plant soft rot.

[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 solution, the nucleotide sequence of the IbNFYA3 gene of the present invention can be a nucleic acid molecule with a coding region of SEQ ID NO.2; can be a DNA molecule that hybridizes with a nucleic acid molecule that is consistent with SEQ ID NO.2 or has a coding region of SEQ ID NO.2 under strict conditions and encodes the protein IbNFYA3; can also be a DNA molecule that has 75% or more homology with a nucleic acid molecule that is consistent with SEQ ID NO.2 or has a coding region of SEQ ID NO.2 and encodes the IbNFYA3 protein. The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; or can be RNA, such as mRNA or hnRNA.

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

[0012] The term "homology" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Homology" includes nucleotide sequences that have 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology to the nucleotide sequence shown in SEQ ID NO.2 of the present invention. Homology can be evaluated by the naked eye or by 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, which comprises the above-mentioned IbNFYA3 gene.

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

[0015] As an additional embodiment, the biological material of the present invention also includes an expression cassette and a transgenic plant cell line.

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

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

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

[0019] Inserting the IbNFYA3 gene into the multiple cloning site of the vector pCAMBIA1300 to obtain the recombinant expression vector pCAMBIA1300-IbNFYA3-GFP;

[0020] Further preferably, the pCAMBIA1300 vector is double-digested with HindIII and EcoRI to recover the large vector fragment, and the target gene sequence is amplified using forward and reverse primers of the IbNFYA3 gene with restriction endonucleases HindIII and EcoRI, and the recovered large vector fragment is connected 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 be specifically 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 IbNFYA3 gene (i.e., the gene encoding the IbNFYA3 protein) into the recipient plant, but also its progeny; for transgenic plants, the gene can be propagated in the species, and the gene can also be transferred into other varieties of the same species using conventional breeding techniques, especially including commercial varieties. The transgenic plants include seeds, callus tissues, complete plants and cells.

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

[0024] Further preferably, the regulating the plant's resistance to soft rot may be increasing the plant's resistance to soft rot or decreasing the plant's resistance to soft rot.

[0025] Further preferably, the disease to 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-mentioned IbNFYA3 protein, the above-mentioned IbNFYA3 gene or the above-mentioned biological material in improving plant disease resistance.

[0028] Preferably, the disease to 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 improving plant disease resistance, comprising the step of overexpressing the IbNFYA3 gene in the plant; the nucleotide sequence of the IbNFYA3 gene is as shown in SEQ ID NO.2.

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

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

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

[0034] Further preferably, the change in soft rot resistance may be an increase in soft rot resistance or a decrease in soft rot resistance.

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

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

[0037] In the above-mentioned method for cultivating transgenic plants, the "introducing a nucleic acid molecule encoding the protein IbNFYA3 into the 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; the recombinant expression vector can specifically be the recombinant plasmid pCB-IbNFYA3.

[0038] Further preferably, the disease to which the disease resistance is directed is soft rot;

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

[0040] The present invention provides the use of the above-mentioned IbNFYA3 protein, the above-mentioned IbNFYA3 gene or the above-mentioned biological material in cultivating highly disease-resistant transgenic plants.

[0041] Further preferably, the disease to which the disease resistance is directed is soft rot;

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

[0043] The present invention provides a method for cultivating highly disease-resistant transgenic plants, comprising the step of overexpressing the IbNFYA3 gene in the plant to obtain the highly disease-resistant transgenic plant; the nucleotide sequence of the IbNFYA3 gene is as SEQ ID NO.2.

[0044] Further preferably, the disease to which the disease resistance is directed is soft rot;

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

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

[0047] The present invention first discovered that overexpressing the NFYA gene in plants can significantly increase the jasmonic acid content and aspartic protease content, thereby effectively resisting soft rot bacteria. Specifically, the present invention provides an IbNFYA3 gene encoding a NFYA transcription factor (protein IbNFYA3), and the gene is introduced into sweet potatoes to obtain transgenic sweet potato plants that overexpress the IbNFYA3 gene. The transgenic tubers are subjected to soft rot resistance identification, and it is found that compared with the wild type, the transgenic tubers have significantly enhanced resistance to soft rot. 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 highly disease-resistant transgenic plants; in short, the IbNFYA3 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

[0048] 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.

[0049] Figure 1 PCR detection of transgenic plants; A is callus cultured in MS solid medium containing 2 mg / L 2,4-D; B is embryonic suspension cells cultured in MS liquid medium containing 2 mg / L 2,4-D; C is embryonic 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 embryonic callus with Hyg resistance cultured in MS solid plate containing 1 mg / LABA and 100 mg / L CS is a pseudo-transgenic plant regenerated in MS solid medium; E is a transgenic IbNFYA3 sweet potato plant subcultured on MS solid medium; F is the phenotype of transgenic sweet potato leaves overexpressing the IbNFYA3 gene; G is the phenotype of transgenic sweet potato roots overexpressing the IbNFYA3 gene; H is the PCR test results of different plants, lane M is Maker, lane W is the negative control water, lane P is the positive control (recombinant plasmid pCAMBIA1300-IbNFYA3), lane WT is a wild-type sweet potato plant, 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 This is the identification of the soft rot resistance of transgenic IbNFYA3 sweet potato tubers; A is the disease picture after the slices were inoculated with soft rot fungi; B is the statistical chart of the disease diameter. DETAILED DESCRIPTION

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] The words “include,” “including,” “have,” “contain,” etc. used in this document are 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] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0058] Example 1 Obtaining and functional verification of proteins and their encoding genes related to sweet potato soft rot

[0059] 1. Obtaining proteins and their encoding genes related to sweet potato soft rot

[0060] 1. Extraction of total RNA from sweet potato: Grind 1 g of young leaves of the sweet potato variety Nong Da Bai (NDB) into powder in liquid nitrogen, add them into a 2 mL centrifuge tube, extract total RNA from sweet potato using the Trizon method, and reverse transcribe the first-strand cDNA using the PrimeScriptTMRT reagent Kitwith gDNAEraser kit.

[0061] 2. With the help of the sweet potato stress resistance gene IbJAZ10 that has been studied in the laboratory, the interactive gene IbNFYA3 was screened through the sweet potato yeast two-hybrid library, and it was compared 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 sequence is:

[0062] IbNFYA3-F: 5'-ATGCTAAATTTCCTCATTCTCCAAG-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 IbNFYA3-F and IbNFYA3-R synthesized in step 2 as primers, PCR amplification was performed to obtain a PCR amplification fragment product of about 990 bp and sequenced.

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

[0066] 2. Application of IbNFYA3 protein in regulating sweet potato soft rot resistance

[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, and the forward and reverse primers were introduced into BglII and PmlI restriction sites, respectively. The primer sequences are as follows:

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

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

[0071] Using the artificially synthesized SEQ ID NO.2 as a template, after PCR amplification, the product was connected to the pMD19-T vector, named pMD-IbNFYA3 vector, and M13-F / R sequencing was performed to ensure the correct nucleotide reading frame and restriction site of the sweet potato IbNFYA3 gene.

[0072] 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 expression vector pCBGUS.

[0073] The vector pCBGUS was digested with BglII and PmlI, and the large vector fragment was recovered. At the same time, the fragment was amplified from the vector pMD-IbNFYA3 using primers IbNFYA3-FF-BglII and IbNFYA3-RR-PmlI, and a fragment of about 990 bp was recovered. The recovered large vector fragment was connected with the fragment of about 990 bp to obtain the target plasmid. The target plasmid was transformed into Escherichia coli DH5a, cultured at 37°C for 20 hours, and the recombinant expression 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 recombinant expression vector pCBGUS, indicating that the recombinant expression vector was constructed correctly, 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, the nucleotide sequence of which includes a CaMV35S promoter, a gene encoding IbNFYA3 protein and a NOS terminator.

[0075] 2. Plant expression vector transformation into Agrobacterium

[0076] Process such as Figure 1 As shown in AG, the details are as follows:

[0077] (1) Thaw the prepared Agrobacterium EHA105 competent cells on ice, add 2 μg of the extracted pCAMBIA1300-IbNFYA3 plasmid, flick the tube wall to mix, and place on ice for 10 min;

[0078] (2) Quick freezing in liquid nitrogen for 5 min, 37°C water bath for 10 min, and ice bath for 5 min;

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

[0080] (4) Spread 200 μL of bacterial solution on LB solid medium containing 100 μg / mL ampicillin and 100 μg / mL rifampicin;

[0081] (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 liquid introduced with the pCAMBIA1300-IbNFYA3 vector. The recombinant Agrobacterium was named EHA105 / pCAMBIA1300-IbNFYA3.

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

[0083] EHA105 / pCAMBIA1300-IbNFYA3 was introduced into sweet potato chestnut fragrance by Agrobacterium-mediated method. The specific method is as follows:

[0084] 6.1) 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 3 times to remove any ethanol and sodium hypochlorite solution that may remain on the stem tips.

[0085] 6.2) 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 stereomicroscope (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 mg / L 2,4-D MS solid medium, and culture in the dark to induce embryonic callus ( Figure 1 (A).

[0086] 6.3) 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 2mg / L 2,4-D, and broken into small cell clusters with tweezers. Culture 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).

[0087] 6.4) Soak the pretreated embryonic suspension cells in the Agrobacterium tumefaciens culture solution, shake gently and let it stand for about 5 minutes, aspirate the Agrobacterium culture solution 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 100 mg / L CS, 11 mg / L Hyg and 2 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).

[0088] 6.5) 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 the 20-mesh sieve but cannot pass through the 30-mesh sieve as transformation materials. Immerse the flask containing about 300 cell clusters in an ultrasonic cleaner (50w, 42KHz) for about 1 minute, then wash once with liquid 2,4-D culture medium, and culture on a shaker for 3 days.

[0089] 6.6) After co-culture, transfer the embryonic cell mass into MS liquid medium containing 1 mg / LABA and 100 mg / L cefotaxime sodium (CS) and wash it three times to try 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 1 mg / LABA and 100 mg / L CS and delay the culture for 1 week. If the medium becomes turbid around 3 days in the middle, replace the medium once.

[0090] 6.7) After delayed culture, the culture medium in the flask was removed as clean as possible, and the cell mass was gently transferred and spread on the filter paper on the MS solid culture medium containing 1 mg / LABA, 100 mg / L CS and antibiotics for vector selection markers, and spread evenly (density is about 50 embryonic suspension cells per plate) so that each cell mass can 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 and hard texture was transferred to the surface of a single filter paper on the MS solid culture medium containing 1 mg / LABA, 100 mg / L CS and antibiotics for vector selection markers for continued selection culture, and then the selection culture medium was replaced every 2 weeks.

[0091] 6.8) After culturing embryonic suspension cells on the selective medium for 8 weeks, select embryonic cells with good growth status and transfer them to MS liquid culture medium containing 1mg / LABA and 100mg / L CS for cultivation. The light and temperature conditions in the culture room are constant: 28℃, 3000Lx, 13h / d. Green mature somatic embryos can be obtained after 3-4 weeks of cultivation.

[0092] 6.9) After 2-4 weeks of induction, the mature somatic embryos that turned green on the ABA medium were transferred to MSMS solid medium containing 1 mg / L ABA and 100 mg / L CS, and cultured at 27±1°C, 13 h of light per day, and a light intensity of 3000 Lx for 4-8 weeks until complete transgenic plants were regenerated ( Figure 1 D in the figure). The proposed transgenic plants were subcultured on MS solid medium under a sterile environment and numbered sequentially. The proposed transgenic plants were propagated once every 6-8 weeks ( Figure 1 E), a transgenic sweet potato overexpressing the IbNFYA3 gene was obtained. The leaf phenotype and tuber phenotype of the transgenic sweet potato overexpressing the IbNFYA3 gene are as follows Figure 1 As shown in F and G. 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.

[0093] (7) The genomic DNA of the leaves of the transgenic plants overexpressing the IbNFYA3 gene was extracted by the CTAB method, and the extracted genomic DNA was used as a template, water and wild-type plants were used as negative controls, and plasmid pCB-IbNFYA3 was used as a positive control. PCR amplification was performed using IbNFYA3-FF-BglII and IbNFYA3-RR-PmlI as primers to obtain a PCR amplification product; if the PCR amplification product contains a band of about 1000 bp, the corresponding sweet potato pseudo-transgenic plant is a sweet potato transgenic positive plant, that is, a transgenic sweet potato overexpressing the IbNFYA3 gene.

[0094] The amplification results were detected by electrophoresis. Figure 1 As shown in H in FIG. As can be seen from the figure, the target band of about 1000 bp was amplified in lanes L1-L7 and the positive control, indicating that the IbNFYA3 gene has been integrated into the sweet potato genome, and proving that these regenerated plants are transgenic plants overexpressing the IbNFYA3 gene. The IbNFYA3 gene in the transgenic plants overexpressing the IbNFYA3 gene was detected, and the results showed that the expression level of the IbNFYA3 gene in the transgenic plants overexpressing the IbNFYA3 gene was higher than that in the wild-type sweet potato, and the expression level of the IbNFYA3 gene in the transgenic plants numbered OE-N11 and OE-N12 was the highest ( Figure 1 I); thereafter, the transgenic sweet potato plants identified as overexpressing the IbNFYA3 gene were propagated and identified for resistance to soft rot.

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

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

[0097] (1) Preparation of bacteria: Soft rot bacteria were cultured in three culture dishes containing PDB medium for 3 days. The surface hyphae were scraped off with an inoculation needle and placed in a conical flask containing 20 mL of PDB medium. A PDB solid culture medium disc with a diameter of 1 cm was then added to the conical flask. The dish was shaken at 60 rpm for 2 h to allow a certain amount of bacteria to be stained on the disc for later use.

[0098] (2) Preparation of potato tubers: Wash the potato tubers, dry them, cut them into slices about 1 cm thick, place them on a sterilized plate, and spray them with sterile water to keep them moist;

[0099] (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;

[0100] (4) Disease evaluation: The diameter of the diseased potato chips was measured 16 hours after inoculation. Figure 2 The results showed that: the experiment proved that overexpression of IbNFYA3 gene in sweet potato improved 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 soft rot resistance.

[0102] 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. 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 biomaterial, characterized in that: The biological material includes the IbNFYA3 gene according to claim 2.

4. The biomaterial according to claim 3, characterized in that The biological materials include recombinant expression vectors and recombinant microorganisms.

5. Use of the IbNFYA3 protein according to claim 1, the IbNFYA3 gene according to claim 2, or the biological material according to claim 3 or 4 in improving plant disease resistance.

6. The use according to claim 5, characterized in that: The disease to which the disease resistance is directed is soft rot; And / or, the plant is sweet potato.

7. A method for improving plant disease resistance, characterized in that: The method comprises the step of overexpressing the IbNFYA3 gene in the plant; the nucleotide sequence of the IbNFYA3 gene is as shown in SEQ ID NO.

2.

8. The method according to claim 7, characterized in that The disease to which the disease resistance is directed is soft rot; And / or, the plant is sweet potato.

9. Use of the IbNFYA3 protein according to claim 1, the IbNFYA3 gene according to claim 2, or the biological material according to claim 3 or 4 in cultivating highly disease-resistant transgenic plants.

10. A method for cultivating highly disease-resistant transgenic plants, characterized in that: The method comprises the steps of overexpressing the IbNFYA3 gene in the plant to obtain the highly disease-resistant transgenic plant; the nucleotide sequence of the IbNFYA3 gene is as shown in SEQ ID NO.2.

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