A method for resisting Fusarium oxysporum

Abstract

The present invention relates to a method for resisting Fusarium oxysporum. The method for resisting Fusarium oxysporum comprises the following steps: using wax gourd-like sweet protein to resist Fusarium oxysporum; the amino acid sequence of the wax gourd-like sweet protein comprises the amino acid sequence shown in SEQ ID NO: 1. In the method provided by the present invention, wax gourd-like sweet protein is used to inhibit the growth of plant pathogenic fungus Fusarium oxysporum, has good antifungal activity, can be used for biological control of plant pathogenic fungi in agriculture, and can achieve considerable economic benefits while solving practical problems.

Description

[0001] This invention is a divisional application. The original Chinese patent application number is: 202410116362.0, the application date is: January 29, 2024, and the patent name at the time of application is: A wax gourd sweet protein, gene and its application. Technical Field

[0002] The invention belongs to the field of applied industrial microorganisms, and in particular relates to a method for resisting Fusarium oxysporum. Background Art

[0003] Thaumatin-like proteins are a class of proteins with structures similar to thaumatin, belonging to pathogenesis-related gene family 5. Thaumatin-like proteins (TLPs) are a highly complex protein family that is related to host defense and developmental processes in plants, animals and fungi. Thaumatin-like proteins are widely available in nature, mainly in plants, animals and fungi. The number and variety of thaumatin-like proteins in plants are rich. For example, there are 106 thaumatin-like proteins in corn, 97 in cotton, 78 in rice, 66 in tobacco, 66 in walnut, 51 in Arabidopsis, and about 59 in poplar.

[0004] Studies have shown that some TLPs proteins have potential value in constructing stress-resistant transgenic plants. Sripriya et al. found that transgenic rice lines expressing a combination of rice chitinase (chi11) and tobacco osmotic protein (ap24) in the same T-DNA showed higher levels of resistance to sheath blight than those expressing chi11 or ap24 alone. Misra et al. ectopically expressed the sweet protein ObTLP1 in basil in Arabidopsis, enhancing resistance to fungal pathogens and tolerance to abiotic stresses (drought and high salinity). Transgenic rapeseed lines obtained by Agrobacterium transformation of the cDNA of the sweet protein Hv-TLP8 isolated from barley increased resistance to the soil pathogen Plasmodium campestris (protozoa). Transgenic tobacco plants overexpressing GbTLP1 (a sweet protein from cotton) showed resistance to different stress factors, especially resistance to the fungus Verticillium dahliae, and tolerance to salinity and drought. In another study, heterologous expression of a TLP isolated from tea plant (CsTLP) conferred resistance to the fungi Phytophthora infestans and Phytophthora infestans in potato plants in potato plants. Therefore, the development of new antifungal proteins would be beneficial for biocontrol.

[0005] Plant fungal diseases are an important factor leading to the reduction in the quality and quantity of crops worldwide. In the process of fungal infection of plants, fungi will also secrete toxins and harmful secondary metabolites, which seriously threaten the safety of agricultural products. The five major food crops, wheat, corn, rice, soybeans, and potatoes, lose a total of 125 million tons of production each year due to fungal diseases. The damage to wheat, corn, and rice alone causes an economic loss of US$60 billion to agriculture worldwide each year. At present, the prevention and control of fungal diseases mainly relies on chemical control, and the use of a large number of chemical agents will cause environmental pollution and pesticide residues. Therefore, green biological control is a way to solve these problems, and choosing appropriate antifungal proteins is an effective means of green biological control.

[0006] Fusarium oxysporum belongs to the class Imperfecti fungi, the order Moniliales, the family Tuberculariaceae, and the genus Fusarium. Fusarium oxysporum is a soil-borne pathogenic fungus with a worldwide distribution and a wide host range. It can cause wilt diseases in more than 100 plant species, including melons, solanaceae, bananas, cotton, legumes, and flowers. Currently, biocontrol agents for Fusarium oxysporum include fungi, bacteria, plant extracts, and actinomycetes. There are no reports on the biological control of Fusarium oxysporum by Benincasa hispida thaumatin-like protein. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention discloses a wax gourd-like sweet protein gene, wax gourd-like sweet protein, an engineered bacterium containing the gene, and an antifungal method and application.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] The present invention provides a wax gourd-like sweet protein, comprising an amino acid sequence shown in SEQ ID NO:1.

[0010] The protein provided by the present invention can inhibit the growth of plant pathogenic fungi Fusarium oxysporum and exhibit good antifungal activity. Genes can be used to produce protein preparations, which can be widely used in the biological control of plant pathogenic fungi in agriculture, and can achieve considerable economic benefits while solving practical problems.

[0011] The present invention provides a wax gourd-like sweet protein gene encoding the wax gourd-like sweet protein.

[0012] The wax gourd sweet protein gene may include the nucleotide sequence shown in SEQ ID NO: 2. The full length of the gene (from the start codon to the stop codon) is 681 bp, G+C% is 53.3%, encoding 226 amino acids, and the theoretical molecular weight of the protein is 25.1 kDa.

[0013] The wax gourd-like sweet protein gene may also include an optimized wax gourd-like sweet protein gene sequence, and the optimized wax gourd-like sweet protein gene includes the nucleotide sequence shown in SEQ ID NO:3.

[0014] The wax gourd-like sweet protein gene is discovered for the first time by the present invention, and the gene can encode a sweet protein-like protein.

[0015] The present invention provides a vector comprising the above-mentioned wax gourd-like sweet protein gene. The vector may be a cloning vector or an expression vector. For example, it may be a recombinant vector. Preferably, the above-mentioned wax gourd-like sweet protein gene may be cloned into pPICZαA.

[0016] The present invention provides a microorganism comprising the above-mentioned wax gourd-like sweet protein gene, wherein the microorganism can carry the wax gourd-like sweet protein gene in various ways, including but not limited to, carrying in the form of a plasmid, carrying on the genome, etc. For example, it can be a recombinant microorganism containing the above-mentioned recombinant vector; preferably, Pichia pastoris X-33 can be used as a host bacteria. The Pichia pastoris engineered strain constructed using the gene can efficiently express wax gourd-like sweet protein.

[0017] The present invention provides the use of one or more of the above wax gourd sweet protein, wax gourd sweet protein gene, vector, and microorganism in antibacterial applications. For example, it can be used to resist plant pathogens; it can be used to inhibit the growth of plant pathogenic fungi; and it can be used to prepare protein preparations.

[0018] Specifically, the present invention provides the use of the above-mentioned wax gourd-like sweet protein gene in resistance to Fusarium oxysporum, wherein the wax gourd-like sweet protein gene encodes wax gourd-like sweet protein, the wax gourd-like sweet protein gene includes the nucleotide sequence shown in SEQ ID NO:3, and the amino acid sequence of the wax gourd-like sweet protein includes the amino acid sequence shown in SEQ ID NO:1.

[0019] Specifically, the present invention provides the use of a vector comprising a wax gourd-like sweet protein gene in resistance to Fusarium oxysporum, wherein the wax gourd-like sweet protein gene encodes wax gourd-like sweet protein, the wax gourd-like sweet protein gene comprises the nucleotide sequence shown in SEQ ID NO:3, and the amino acid sequence of the wax gourd-like sweet protein comprises the amino acid sequence shown in SEQ ID NO:1.

[0020] Furthermore, the vector may be obtained by cloning the wax gourd-like sweet protein gene into pPICZαA.

[0021] Specifically, the present invention provides the use of a microorganism comprising a wax gourd-like sweet protein gene in resistance to Fusarium oxysporum, wherein the wax gourd-like sweet protein gene encodes wax gourd-like sweet protein, the wax gourd-like sweet protein gene comprises the nucleotide sequence shown in SEQ ID NO:3, and the amino acid sequence of the wax gourd-like sweet protein comprises the amino acid sequence shown in SEQ ID NO:1.

[0022] Furthermore, the microorganism can use Pichia pastoris X-33 as a host strain.

[0023] The wax gourd sweet protein of the present invention can be used as an antibacterial protein in agricultural applications against plant pathogenic fungi.

[0024] In the above application, the plant pathogen may be Fusarium oxysporum. The vector may be obtained by cloning the above wax gourd-like sweet protein gene into pPICZαA. The microorganism may use Pichia pastoris X-33 as a host. The wax gourd-like sweet protein gene may include the nucleotide sequence and / or the optimized sequence shown in SEQ ID NO:2. The optimized sequence may include the nucleotide sequence shown in SEQ ID NO:3.

[0025] The wax gourd-like sweet protein gene provided by the invention has an upregulated expression in wax gourd root tissue induced by wilt pathogen; the wax gourd-like sweet protein provided by the invention has the activity of inhibiting spore formation of wilt pathogen.

[0026] The present invention also provides a method for resisting Fusarium oxysporum, comprising the following steps: using the above-mentioned wax gourd-like sweet protein to resist Fusarium oxysporum; the amino acid sequence of the wax gourd-like sweet protein includes the amino acid sequence shown in SEQ ID NO:1.

[0027] The method for preparing wax gourd-like sweet protein may include the following steps: constructing a recombinant vector containing a wax gourd-like sweet protein gene, wherein the wax gourd-like sweet protein gene encodes wax gourd-like sweet protein; the wax gourd-like sweet protein gene includes the nucleotide sequence shown in SEQ ID NO:3; using Pichia pastoris as a host bacteria to construct a recombinant strain expressing the wax gourd-like sweet protein gene, and inducing expression using methanol to obtain the wax gourd-like sweet protein.

[0028] Furthermore, the recombinant vector can use pPICZαA as a skeleton; and the host bacteria is Pichia pastoris X-33.

[0029] The present invention provides a theoretical basis for the research and development of key technologies for the prevention and control of plant fungal diseases by screening antifungal plant genes and the proteins they encode and revealing the mechanism of action of antifungal proteins. It also applies them to biological control, providing technical support for the green and high-quality production of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram showing the strategy for cloning sweet protein genes in wax gourd.

[0031] Figure 2 This is a diagram of the experimental scheme for the efficient expression of wax gourd sweet protein genes in P. pastoris X-33 (pPICZαA).

[0032] Figure 3 The results of colony PCR verification; where: M: DL5000 nucleic acid marker; 1-8: 8 clones randomly selected from the plate.

[0033] Figure 4 The figure above is the result of WB staining to detect the expression of the target protein. The upper figure is the result of staining, in which M: Marker, 1-7: target protein (clone 1 to clone 7), 8: positive control. The lower figure is the result of WB experiment, in which 1-7: are the target proteins (clone 1 to clone 7) respectively.

[0034] Figure 5 This is the experimental result of protein amplification expression detection, where M: Marker, 1: target protein, 2: positive control.

[0035] Figure 6 It is the result of purified protein detection, where M: Marker, 1: BhTLP22 effluent, 2: washing solution, 3: purified protein.

[0036] Figure 7 The standard curve for protein concentration determination.

[0037] Figure 8 Expression of BhTLP1 and BhTLP22 in root tissue of wax gourd induced by Fusarium oxysporum.

[0038] Fig. 9 These are the experimental results of the effects of recombinant wax gourd-like sweet proteins on Fusarium oxysporum, where: control (CK): Elutionbuffer; treatment group (BhTLP22-treated): addition of recombinant wax gourd-like sweet protein BhTLP22; ad are plate photos of Fusarium oxysporum growth (cultured for 8 days); e and f are the effects on Fusarium oxysporum cells (cultured for 8 days); g is a curve graph of Fusarium oxysporum diameter, and the horizontal axis dpi is the abbreviation of days post-inoculation. DETAILED DESCRIPTION

[0039] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0040] The present invention uses the transcriptome data of the variant of winter melon, the winter melon, and the genome sequence information of winter melon to successfully obtain the sequence of the gene of Benincasa hispida thaumatin-like protein through PCR amplification. The full length of the gene (from the start codon to the stop codon) is 681 bp, the G+C% is 53.3%, and it encodes 226 amino acids.

[0041] The amino acid sequence of wax gourd sweet protein is shown in SEQ ID NO: 1:

[0042] MGISNICFLFFLALLSFYSQTHAATFQVRNNCPFTVWAAAVPGGGRRLNRNDVWTFNVNPGTVAARIWPRTNCNFDGSGRGRCQTGDCGGLLQCQAYGTPPNTLAEYALNQFN NLDFFDISLVDGFNVPMEFSPTSGGCTRGIRCTADINGQCPNELRAPGGCNNPCTVFGGDRYCCTAPNSSCGPTDYSRFFKNRCPDAYSYPKDDATSTFTCPGGTHYRVVFCP

[0043] The full-length nucleotide sequence of the wax gourd sweet protein gene is shown in SEQ ID NO: 2:

[0044] ATGGGTATCTCTAATATTTGTTTTCTCTTCTTTCTTGCTCTTCTTTCTTTCTATTCCCAAACCCATGCAGCTACTTTTCAAGTCCGAAACAACTGCCCCTTCACTGTTTGGGCTGCTGCAGTACCCGGCGGTGGACGACGACTTAATCGAAACGATGTTTGGACATTTAA CGTGAATCCGGGCACCGTTGCTGCTCGTATTTGGCCTCGAACTAACTGCAACTTCGACGGTTCTGGCCGGGGTAGATGCCAGACTGGCGACTGTGGTGGTCTCCTCCAGTGCCAAGCTTACGGCACTCCGCCGAACACCCTTGCCGAATACGCGTTAAATCAGTTCAATA ACTTGGACTTCTTCGATATCTCTCTCGTCGATGGATTTAACGTTCCGATGGAGTTCAGCCCGACTTCTGGGGGGTGCACTCGTGGCATCAGGTGCACCGCGGACATCAACGGACAGTGCCCGAACGAGCTTAGGGCTCCTGGAGGGTGCAACAACCCGTGCACCGTGTTC GGGGGTGATCGATACTGCTGCACCGCCCCCAACAGCAGCTGTGGCCCGACGGATTACTCTAGGTTCTTCAAGAATCGGTGCCCAGATGCATACAGTTACCCAAAAGACGATGCAACCAGCACATTCACTTGCCCCGGGGGAACCCACTACAGGGTTGTCTTCTGTCCTTGA

[0045] When cloning the gene of winter melon sweet protein, Pichia pastoris can be used as the engineering strain, such as Figure 1 As shown, the method comprises the following steps: removing the signal peptide and codon optimization of the wax gourd sweet protein gene, synthesizing the optimized target gene into pPICZαA by gene synthesis technology, the target gene is adjacent to the vector α-factor, and the C-terminus is provided with 6×His to obtain the target vector. The target vector is linearized and purified and recovered, and yeast P. pastoris X-33 electrotransformation competent state is prepared, electrotransformed, transformants are screened, and methanol is used to induce expression. Through the above method, wax gourd sweet protein is efficiently expressed using Pichia pastoris as an engineering strain, and the induced expression amount can be 148ug / ml.

[0046] The engineered strain constructed using this gene can efficiently express wax gourd-like sweet protein. When the wild type WT of Fusarium oxysporum is used as the target, the growth of Fusarium oxysporum is significantly inhibited after being cultured on a medium supplemented with the recombinant wax gourd-like sweet protein BhTLP22. Microscopic observation shows that the death of Fusarium oxysporum in the treatment group is more serious than that in the control group. This shows that the protein can inhibit the growth of plant pathogenic fungus Fusarium oxysporum and exhibits good antifungal activity. The protein preparation produced using the gene can be used for biological control of plant pathogenic fungi in agriculture, which can solve practical problems and achieve considerable economic benefits.

[0047] In the present invention, unless otherwise specified, the experimental methods used are all conventional experimental methods in the art; the materials, reagents, and instruments used are all conventional materials, reagents, and instruments in the art, which can be obtained through commercial channels or prepared by conventional methods in the art.

[0048] pPICZαA was purchased from Nanjing Ruiyuan Biotechnology Co., Ltd.; the host strain P. pastoris X-33 was purchased from Nanjing Ruiyuan Biotechnology Co., Ltd.; and Fusarium oxysporum was purchased from the Treasure Center of the Chinese Culture Resource Bank.

[0049] Buffer A was prepared according to the following ratio: 20 ml YPD + 2 ml 2 M 4-hydroxyethylpiperazineethanesulfonic acid HEPES (pH=8.0, filter sterilized) + 0.5 ml 1 M dithiothreitol DTT (filter sterilized).

[0050] Lysis buffer formula includes: prepared with water, 50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH=8.0, filtered and sterilized.

[0051] Wash buffer formula includes: prepared with water, 50mM NaH2PO4, 300mM NaCl, 20mM imidazole, pH=8.0, filtered and sterilized.

[0052] The elution buffer formula includes: prepared with water, 50mM NaH2PO4, 300mM NaCl, 250mM Mimidazole, pH=8.0, filtered and sterilized.

[0053] YPD: 1% yeast extract, 2% peptone, 2% glucose. If preparing solid culture medium, add 2% agar powder. All the above are weight percentages.

[0054] YPG medium: 1% yeast extract, 2% peptone, 3% ethanol and 3% glycerol, all are mass percentages. If solid culture medium is prepared, add 2% agar powder, all the above are mass percentages.

[0055] The preparation method of PDA culture medium can be prepared according to the following proportions: 200g of peeled potatoes are cut into pieces, added with 800mL of water, boiled for 20 minutes, filtered with gauze, added with 20g of glucose and 15g of agar powder, fixed to 1L, and sterilized with high-pressure steam at 121°C for 20min; after sterilization, the culture medium with the temperature dropped to about 50°C is poured into a sterile culture dish to make a PDA plate.

[0056] Liquid Czapek medium was prepared according to the following proportions: 3 g sodium nitrate, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate (MgSO4·7H2O), 0.5 g potassium chloride, 0.01 g ferrous sulfate, 30 g sucrose, and 1000 mL distilled water.

[0057] Sodium nitrate was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 10019928; potassium dihydrogen phosphate was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: HX10025; MgSO4·7H2O was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: XW221890881; potassium chloride was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 10016308; ferrous sulfate was purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number: T20691; sucrose was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number: 10021487; 10× buffer was purchased from Thermo Fisher Scientific; SacI was purchased from Thermo Fisher Scientific, catalog number: ER1131; nucleic acid precipitation agent was purchased from Beijing Solebow Technology Co., Ltd., catalog number: SA1020; BMMY medium was purchased from Sangon Biotech (Shanghai) Co., Ltd. (abbreviated as: Sangon), catalog number: B540131-0500; protein loading buffer was purchased from Beijing Quanshijin Biotechnology Co., Ltd., with the catalog number DL101-02; ECL was purchased from Bio-Technology, with the catalog number C510045-0100; gravity column was purchased from Beijing Solebeau Technology Co., Ltd., with the catalog number YA2450; Ni-NTA beads were purchased from Beijing Rambolide Biotechnology Co., Ltd., with the catalog number N30210; polysaccharide and polyphenol plant total RNA extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., with the catalog number DP441; SeqHuntFirst Strang Cdna Synthesis Kit was purchased from Entropy Theory (Shenzhen) Biotechnology Co., Ltd.; Bradford protein concentration determination kit was purchased from Huayueyang Biotechnology Co., Ltd., with the catalog number HYJYKY5975; 2x U+SYBR qPCR Master Mix was purchased from Entropy Theory (Beijing) Biotechnology Co., Ltd., with the catalog number BC05-100.

[0058] In each example, for the convenience of description, the wax gourd-like sweet protein gene is referred to as BhTLP22 gene.

[0059] The following is an introduction through specific embodiments.

[0060] Example 1

[0061] like Figure 2 As shown, the efficient expression of wax gourd-like sweet protein gene in Pichia pastoris includes the following steps: first, constructing a recombinant vector including the optimized BhTLP22 gene; second, linearizing the target vector; and finally, electrotransforming into Pichia pastoris X-33 and screening transformants.

[0062] 1.1 Construction of recombinant vector pPICZαA-BhTLP22

[0063] Reference to Benincasa hispida thaumatin-like protein (LOC120067974), which was obtained from the search of NCBI database.

[0064] The CDS region of the gene was codon optimized, and the sequence codons were optimized to be better expressed in yeast. Shanghai Qingke Biotechnology Co., Ltd. used chemical methods to synthesize the above optimized gene sequence, and directly synthesized the optimized gene sequence into pPICZαA. The target gene was next to the α-factor of the vector pPICZαA, and 6×His was attached to the C-terminus. The XhoI / SmaI restriction enzyme digestion method was used to verify and obtain the correct recombinant vector pPICZαA-BhTLP22.

[0065] The optimized gene nucleotide sequence is shown in SEQ ID NO:3:

[0066] GCTACTTTCCAAGTTAGAAACAACTGTCCATTCACTGTTTGGGCTGCTGCTGTTCCAGGTGGTGGTAGAAGATTGAACAGAAACGATGTTTGGACTTTCAACGTTAACCCAGGTACTGTTGCTGCTAGAATCTGGCCAAGAACTAACTGTAA CTTCGATGGTTCTGGTAGAGGTAGATGTCAAACTGGTGATTGTGGTGGTTTGTTGCAATGTCAAGCTTACGGTACTCCACCAAACACTTTGGCTGAATACGCTTTGAACCAATTCAACAACTTGGATTTCTTCGATATCTCTTTTGGTTGATG GTTTCAACGTTCCAATGGAATTCTCTCCAACTTCTGGTGGTTGTACTAGAGGTATCAGATGTACTGCTGATATCAACGGTCAATGTCCAAACGAATTGAGAGCCCCAGGTGGTTGTAACAACCCATGTACTGTTTTCGGTGGTGATAGATAC TGTTGTACTGCTCCAAACTCTTCTTGTGGTCCAACTGATTACTCTAGATTCTTCAAGAACAGATGTCCAGATGCTTACTCTTACCCAAAGGATGATGCTACTTCTACTTTCACTTGTCCAGGTGGTACTCATTACAGAGTTGTTTTCTGTCCA

[0067] 1.2 Linearization of target vector

[0068] The above-constructed recombinant plasmid (pPICZαA-BhTLP22) was linearized using restriction endonuclease SacI, comprising the following steps:

[0069] First, prepare the vector enzyme digestion system, which includes: plasmid (5-10ug) converted into a volume of 6ul, 10× buffer 5ul, SacI 1ul, and use ddH2O to make up to 50ul system.

[0070] Secondly, the enzyme cleavage system was placed at 37°C for 8 hours, and then the enzyme cleavage products were recovered. After the successful enzyme cleavage was detected, the reaction system was inactivated at 65°C for 20 minutes.

[0071] 1.3 Purification and recovery of linearized vector by ethanol precipitation

[0072] The linearized vector is purified and recovered by ethanol precipitation, comprising the following steps:

[0073] (1) Prepare the vector purification system: 50ul of the inactivated enzyme cleavage product (prepared in Example 1.2), 10ul of a nucleic acid precipitation aid, 6ul of 3M NaAc (pH=5.2), and 165ul of anhydrous ethanol;

[0074] (2) The above system was allowed to stand at -20°C for more than 35 min; centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was discarded. At this time, a white precipitate could be observed on the wall; 400 ul of pre-cooled 80% ethanol was added to resuspend the precipitate, and the mixture was centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was discarded. The tube was opened and dried; 10 ul of ddH2O was added to dissolve the precipitate.

[0075] 1.4 Preparation of competent yeast for electroporation

[0076] The preparation method of yeast electroporation competent cells includes the following steps: adding 5 ml YPD to a 50 ml centrifuge tube, inoculating P. pastoris X-33 strain, and culturing at 30°C overnight; at about 1 pm the next day, transferring 50 ul of bacterial solution to a 250 ml conical flask containing 50 ml YPD, and culturing overnight until OD 600 =1.3-1.5; centrifuge at 4°C, 4000rpm for 5min, resuspend with 10ml buffer A, place in a 30°C water bath for 15min, add pre-cooled sterile water to 50ml. Centrifuge at 4°C, 4000rpm for 5min, resuspend with 50ml pre-cooled resuspension solution, the resuspension solution is prepared according to the following ratio: 50ml sterile water + 0.3ml 2M HEPES, pH=8.0; centrifuge at 4°C, 4000rpm for 5min, resuspend with 4ml pre-cooled 1M sorbitol; centrifuge at 4°C, 4000rpm for 5min, resuspend with 100ul pre-cooled 1M sorbitol (the bacterial solution is viscous at this time), dispense into 80ul / tube, and place on ice.

[0077] 1.5 Electroporation of linearized vector into Pichia pastoris

[0078] The recovered and purified linearized vector was electroporated into Pichia pastoris, comprising the following steps: taking 80ul P. pastoris X-33 competent cells (prepared by the method of Example 1.4), adding 6ug linearized pPicZαA-BhTLP22 (prepared by the method of Example 1.3), mixing and transferring to a pre-cooled 0.2cm electroporation cup; placing on ice for 5min; setting the electroporation parameters for Pichia pastoris (1.5kV, 25uF, 200Ω), and electroporating; immediately adding 2ml of pre-cooling solution, the pre-cooling solution was prepared according to the following ratio: 10ml1M sorbitol+100ul 2M HEPES, pH = 8.0; transfer to a 2 ml sterile centrifuge tube; incubate at 30°C for 1-2 h; dilute 5-fold, 10-fold, and 100-fold, respectively, and spread 300 ul / plate on a YPD plate (15 cm in diameter) containing 100 mg / l Zeocin, and culture at 30°C until clones grow.

[0079] 1.6 Screening of transformants

[0080] Screening transformants includes the following steps: randomly selecting 8 clones from the plate and performing colony PCR;

[0081] The sequence of the forward primer used in the colony PCR process is shown in SEQ ID NO:4: CAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCGAGCGCCGCCATG.

[0082] The sequence of the reverse primer used in the colony PCR process is shown in SEQ ID NO: 5: CACGATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCTACGATT.

[0083] The colony PCR used the rapid amplification yeast cloning kit (Cat. No. RY8001) independently developed by Nanjing Ruiyuan Biotechnology Co., Ltd. The colony PCR product was subjected to electrophoresis. The experimental results are as follows Figure 3 As shown, it can be seen that the size of the electrophoresis band is consistent with the expectation; 2 of them are selected for sequencing, and after the alignment is correct, they are retained for later use. The above method obtains the Pichia pastoris recombinant strain containing BhTLP22.

[0084] Example 2 High-efficiency expression of wax gourd-like sweet protein gene in Pichia pastoris

[0085] 2.1 Transformant expression test

[0086] The transformant expression test comprises the following steps: selecting 7 clones of the above-mentioned correctly verified Pichia recombinant strain containing BhTLP22 (prepared by the method of Example 1, respectively named as clone 1 to clone 7) and inoculating them into 50 ml conical flasks, each conical flask is filled with 5 ml YPG medium, and cultured at 30°C and 220 rpm for 1-2 days until the bacterial solution is saturated; transferring to a 50 ml centrifuge tube, centrifuging at 4000 rpm for 5 min, and discarding the supernatant; resuspending the bacteria with 5 ml BMMY medium, transferring to a new sterile 50 ml conical flask, adding methanol to a final concentration of 0.75% (volume percentage), and culturing at 28°C and 220 rpm for 6 days; because methanol is volatile, methanol is added every 24 hours to make the final concentration of methanol 0.75% (volume percentage); collecting the bacterial solution in the morning of the 6th day, centrifuging at 4000 rpm for 5 min, collecting the supernatant, and using it for staining and WB identification to examine the expression.

[0087] The staining and WB identification include the following steps: take 40ul protein solution (i.e., the supernatant prepared by the above method), add appropriate amount of protein loading Buffer, boil at 100℃ for 5-10min; centrifuge at 12000rpm for 2min, draw 20ul for protein electrophoresis, first run the stacking gel at 80V, then 100V until the end of electrophoresis; one gel is used for Coomassie Brilliant Blue staining, and the other is used for membrane transfer, 100V, 90min; after the membrane transfer, wash the PVDF membrane with TBST once, 10min, pour out the TBST; block with TBST containing 5% skim milk powder, block on a shaker at 50rpm at room temperature for 1h; dilute the primary antibody with blocking solution containing 5% skim milk powder, hybridize the membrane on a low-speed shaker at room temperature for 4h: wash the membrane with TBST 3 times, 10min each time; dilute the secondary antibody with blocking solution containing 5% skim milk powder, hybridize the membrane on a low-speed shaker at room temperature for 45min; wash the membrane with TBST 3 times, 10min each time; scan the membrane after ECL exposure.

[0088] The experimental results are as follows Figure 4 As shown in the figure above, the results of the staining of clones 1 to 7 are shown. It is speculated that the staining may be low in sensitivity and lack of specificity, so no bands are seen. However, the absence of bands does not mean that there are no target bands. Therefore, WB experiments were further used to verify whether clones 1 to 7 expressed the target protein. Figure 4 As can be seen in the figure below, clone 2, clone 4, clone 6 and clone 7 were successfully expressed. It is speculated that some clones failed to express successfully due to differences in the expression of the vector after transformation into yeast or experimental operations. Among the clones that can express, clone 7 expressed the most ideally, so clone 7 was selected for subsequent experiments.

[0089] 2.2 Expanded expression of the best transformant

[0090] The optimal transformant (clone 7 in Example 2.1) was expanded for expression, comprising the following steps: adding 50 ml of YPG medium to a 250 ml conical flask, inoculating the optimal expression transformant (clone 7 in Example 2.1), and culturing at 30°C and 220 rpm for 1-2 days until the bacterial solution is saturated; transferring to a 50 ml centrifuge tube, centrifuging at 4000 rpm for 5 min, and discarding the supernatant; resuspending the bacteria with 50 ml of BMMY medium, transferring to a new sterile 250 ml conical flask, adding methanol to a final concentration of 0.5% (volume percentage), and culturing at 28°C and 220 rpm for 6 days; adding methanol every 24 hours to make the final concentration of methanol 0.75% (volume percentage); collecting the bacterial solution on the morning of the 6th day, centrifuging at 4000 rpm for 5 min, and collecting the supernatant for expression identification.

[0091] Expression identification was performed by staining and WB identification, and the specific method was the same as Example 2.1.

[0092] The experimental results are as follows Figure 5 As shown, from Figure 5 As can be seen, the amplification and expression of positive clone 7 was successful.

[0093] 2.3 Protein purification

[0094] The sample for expression identification was subjected to protein purification, comprising the following steps: 5 ml Ni-NTA beads were filled in a 30 ml gravity column; the column was equilibrated with 5 ml Lysis buffer, repeated 3 times; 50 ml protein solution (i.e., BhTLP22 effluent) was passed through the column twice; 10 ml Wash buffer was added for washing, repeated 5 times, and the washing solution was reserved for subsequent SDS-PAGE electrophoresis; 5 ml Elution buffer was added for elution to obtain the purified protein.

[0095] The purified recombinant wax gourd sweet protein was subjected to SDS-PAGE electrophoresis. Figure 6 As shown, from Figure 6 It can be seen that the protein sample was purified successfully without any other impurities.

[0096] The concentration of the purified protein was detected using a Bradford protein concentration determination kit, including the following steps: diluting the BSA standard to 1 mg / ml as needed; making a standard curve, and calculating the concentration of the sample according to the standard curve.

[0097] The preparation of the standard curve includes the following steps: prepare BSA standard solutions of different concentrations, with BSA concentrations of 0ug / ml, 25ug / ml, 50ug / ml, 75ug / ml, and 100ug / ml, respectively; take 20ul of the above solutions and add 180ul of Coomassie Brilliant Blue G250 dye, mix well, and measure the absorbance at 595nm with an enzyme marker. With the absorbance value as the horizontal axis and the BSA concentration (ug / ml) as the vertical axis, draw the standard curve equation, such as Figure 7 shown.

[0098] Take 20ul of the purified protein sample to be tested, add 180ul of Coomassie Brilliant Blue G250 dye, mix well, measure the absorbance at 595nm with an enzyme marker, and calculate the protein concentration based on the detected absorbance value and the standard curve equation. After testing, the protein concentration in the sample is about 148.08μg / ml.

[0099] Example 3 Induction test of wilt pathogen on wax gourd roots

[0100] The experimental method includes the following steps:

[0101] (1) Plate culture of wilt pathogen: Inoculate Fusarium oxysporum onto PDA plates and culture at 26°C in the dark for 5 days before using for liquid culture.

[0102] (2) Liquid culture of Fusarium wilt: Use a sterile toothpick to pick up Fusarium oxysporum on the PDA medium and inoculate it into liquid Czapek medium together with the PDA medium. Cultivate at 26°C and 200 rpm for 3 days. Then collect the spores of Fusarium oxysporum by centrifugation and resuspend them in sterile water.

[0103] (3) Inoculation experiment: Using hydroponic culture to achieve two liquids and one heart of wax gourd seedlings, the prepared bacterial solution was added to the hydroponic box to a concentration of 1×10 6 The root tissues were taken after treatment for 0h, 3h, 12h and 48h respectively, treated with liquid nitrogen and stored at -80℃.

[0104] (4) Extraction of RNA from wax gourd root tissue: RNA was extracted according to the instructions of the polysaccharide and polyphenol plant total RNA extraction kit.

[0105] (5) Synthesizing the first strand cDNA using SeqHunt First Strang Cdna Synthesis Kit, comprising the following steps:

[0106] ①Take template RNA and add primers. The system (total volume 14μl) includes: Total RNA 1μg, Random Primers 1μl, Oligo(dT) 15Primer 1μl, Nuclease-Free Water to 14μl. Pre-denature the mixture of template RNA and primers at 65℃ for 5min, then take out and place on ice to obtain a mixed sample. Random Primers, Oligo(dT) 15 All primers are provided with the SeqHunt First Strang Cdna Synthesis Kit.

[0107] ② Prepare the reaction system. Add 10μl of the mixed sample from step ①, 2μl of 10X Enzyme Mix, and 4μl of 5X Reaction Buffer to each sample tube. Use a pipette to gently blow and mix. Perform the first-strand cDNA synthesis reaction under the following conditions: 25℃, 5min; 50℃, 30min; 85℃, 2min. Obtain cDNA.

[0108] (6) Real-time quantitative reverse transcription PCR (qRT-PCR):

[0109] The cDNA obtained in step (5) was used as a template for real-time quantitative PCR to prepare the reaction system (total volume 20 μl): 2x U + SYBR qPCR Master Mix 10μl, forward primer 0.4μl, reverse primer 0.4μl, template cDNA 2μl, Nuclease-free water to make up to 20μl.

[0110] The forward primer for detecting BhTLP1 is GGCGTTTGCAAATCCGAACA (SEQ ID NO: 6); the reverse primer for detecting BhTLP1 is CTGAAATGTCAGCCGCTTGC (SEQ ID NO: 7).

[0111] The forward primer for detecting BhTLP22 is CGACTGTGGTGGTCTCCTCC (SEQ ID NO: 8); the reverse primer for detecting BhTLP22 is AGTCGGGCTGAACTCCATCG (SEQ ID NO: 9).

[0112] The forward primer of Bh-actin (internal reference) was TACAGGAGGGAGCATGGGATG (SEQ ID NO: 10); the reverse primer of Bh-actin (internal reference) was AAGATGGCCTTCTGCAACTGAG (SEQ ID NO: 11).

[0113] The cycle program of real-time quantitative PCR: ①95℃, 5min; ②95℃, 15sec; ③60℃, 40sec; ④Go to ②, 40cycles; ⑤Melting curve: the program comes with the qPCR instrument.

[0114] The experimental results are as follows Figure 8 As shown, from Figure 8 It can be seen that under the induction of Fusarium oxysporum, compared with BhTLP1, BhTLP22 was significantly upregulated at 3h, 12h, and 48h.

[0115] Example 4 Verification of the Effect of BhTLP22 Protein on the Plate Colonies of Fusarium oxysporum

[0116] The culture medium supplemented with BhTLP22 protein was used as the treatment group, and the plate without BhTLP22 protein was used as the control group.

[0117] In the treatment group, the BhTLP22 protein solution (prepared by the method of Example 2.3) was added to the PDA culture medium to a final concentration of 0.2 mM of the BhTLP22 protein.

[0118] In the control group (CK), an equal amount of Elution buffer was used instead of the BhTLP22 protein solution.

[0119] Fusarium oxysporum was inoculated onto a PDA medium plate, and the inoculated plate was cultured at 26° C. in the dark. The diameters of the colonies after 2, 4, 6, and 8 days of culture were measured.

[0120] Depend on Fig. 9 It can be seen that compared with the control group CK, the colony diameter of Fusarium oxysporum on the plate added with BhTLP22 protein is smaller and grows slower, so BhTLP22 protein has a significant inhibitory effect on the growth of Fusarium oxysporum. BhTLP22 protein can be used to prepare a preparation for preventing and treating Fusarium oxysporum, and can be used to prevent and treat diseases caused by Fusarium oxysporum.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for resisting Fusarium oxysporum, characterized in that: The following steps are involved: The wax gourd-like sweet protein is used to resist Fusarium oxysporum; the amino acid sequence of the wax gourd-like sweet protein is shown in SEQ ID NO: 1; the wax gourd-like sweet protein is prepared by the following method: (1) constructing a recombinant vector containing a wax gourd-like sweet protein gene; the recombinant vector uses pPICZαA as a backbone; the wax gourd-like sweet protein gene encodes wax gourd-like sweet protein; the nucleotide sequence of the wax gourd-like sweet protein gene is shown in SEQ ID NO: 3; (2) Using Pichia pastoris as a host bacterium to construct a recombinant strain expressing a wax gourd-like sweet protein gene, wherein the host bacterium is Pichia pastoris P. pastoris X-33; Use methanol to induce expression to obtain wax gourd-like sweet protein.

2. The use of wax gourd-like sweet protein gene in resistance to Fusarium oxysporum, characterized in that: The wax gourd-like sweet protein gene encodes wax gourd-like sweet protein, and the nucleotide sequence of the wax gourd-like sweet protein gene is shown in SEQ ID NO:

3.

3. Application of a vector comprising a wax gourd sweet protein gene in resistance to Fusarium oxysporum, characterized in that: The wax gourd-like sweet protein gene encodes wax gourd-like sweet protein, and the nucleotide sequence of the wax gourd-like sweet protein gene is shown in SEQ ID NO: 3; the vector is obtained by cloning the wax gourd-like sweet protein gene into pPICZαA.

4. The use of a microorganism comprising a wax gourd-like sweet protein gene in resistance to Fusarium oxysporum, characterized in that: The wax gourd-like sweet protein gene encodes wax gourd-like sweet protein, and the nucleotide sequence of the wax gourd-like sweet protein gene is shown in SEQ ID NO: 3; the microorganism is Pichia pastoris P. pastoris X-33 is the host bacteria.

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