CN117924447B

CN117924447B - White gourd sweet protein, gene and application thereof

Info

Publication number: CN117924447B
Application number: CN202410116362.0A
Authority: CN
Inventors: 杜旋; 刘娜; 王颖
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2024-01-29
Filing date: 2024-01-29
Publication date: 2024-06-18
Anticipated expiration: 2044-01-29
Also published as: CN118388616A; CN117924447A

Abstract

The invention relates to white gourd sweet protein, a gene and application thereof. The sweet melon protein includes the amino acid sequence shown in SEQ ID No. 1. A sweet protein gene of the wax gourd, which codes the sweet protein of the wax gourd. The Pichia pastoris engineering strain constructed by the gene can efficiently express the sweet proteins of the cucurbitaceous plants. The protein can inhibit the growth of plant pathogenic fungi Fusarium oxysporum and has good antifungal activity. The protein preparation produced by the gene can be used for biological control of agricultural plant pathogenic fungi, and can obtain considerable economic benefit while solving the practical problem.

Description

White gourd sweet protein, gene and application thereof

Technical Field

The invention belongs to the field of applied industrial microorganisms, and particularly relates to a novel antifungal protein, namely a sweet melon protein, a gene for encoding the protein, engineering bacteria containing the gene and application of the engineering bacteria in antifungal aspect.

Background

Sweet proteins (thaumatin-like proteins) are a class of proteins with a structure similar to that of sweet protein (Thaumatin), belonging to the disease-associated gene family 5 (Pathogenesis-Related-5). Thaumatin-like proteins (TLPs) are a highly complex family of proteins involved in host defense and developmental processes in plants, animals and fungi. The source of the sweet-like proteins in nature is wide, and the sweet-like proteins mainly exist in plants, animals and fungi, wherein the quantity and the types of the sweet-like proteins in the plants are rich, for example, 106 sweet-like proteins in corn, 97 sweet-like proteins in cotton, 78 sweet-like proteins in rice, 66 sweet-like proteins in tobacco, 66 walnut, 51 Arabidopsis and about 59 sweet-like proteins in poplar.

Studies have shown that a portion of the TLPs have potential value in constructing stress-tolerant transgenic plants. Sripriya et al found that transgenic rice lines expressing a combination of rice chitinase (chi 11) and tobacco penetration protein (ap 24) in the same T-DNA exhibited higher levels of sheath blight resistance than those lines expressing chi11 or ap24 alone. Misra et al ectopic expression of sweet-like protein ObTLP1 in Ocimum basilicum in Arabidopsis, enhanced resistance to fungal pathogens and tolerance to abiotic stresses (drought and high salinity). Transgenic canola lines obtained by Agrobacterium transformation of the cDNA for the sweet-like protein Hv-TLP8 isolated from barley have increased resistance to the soil pathogen P.brassicae (protozoa). Transgenic tobacco plants overexpressing GbTLP1, a cotton sweet-like protein, exhibit resistance to different stress factors, in particular resistance to the fungus verticillium dahliae, resistance to salinity and drought. In another study, heterologous expression of TLP (CsTLP) isolated from tea trees confers resistance to the fungi phytophthora phaseoli and phytophthora solani on potato plants. Therefore, it would be advantageous to have biological control if new antifungal proteins could be developed.

Plant fungal diseases are important factors for reducing the quality and quantity of crops worldwide, and toxins and harmful secondary metabolites can be secreted in the process of infecting plants by fungi, so that the safety of agricultural products is seriously threatened. Five cereal crops of wheat, corn, rice, soybean and potato are reduced by 1.25 hundred million tons per year due to fungal diseases, and only the harm to the wheat, the corn and the rice brings about economic loss of 600 hundred million dollars per year for worldwide agriculture. At present, the means for preventing and controlling fungal diseases mainly depend on chemical prevention and control, and the use of a large amount of chemical agents can cause the problems of environmental pollution and pesticide residues. Green biocontrol is thus one way to solve these problems and the selection of suitable antifungal proteins is an effective means of green biocontrol.

Fusarium oxysporum (F.oxysporum) is a fungus of the genus Fusarium (IMPERFECTI FUNGI), from the order of the Stemonales, from the family Oenotheraceae (Tuberculariaceae), and from the genus Fusarium (Fusarium). Fusarium oxysporum is a soil-borne pathogenic fungus distributed worldwide, has a wide host range, and can cause the occurrence of plant wilt of more than 100 plants such as melons, solanaceae, bananas, cottons, leguminosas, flowers and the like. The biocontrol factors currently used for fusarium oxysporum comprise fungi, bacteria, plant extracts, actinomycetes and the like. Biological control of fusarium oxysporum has not been reported for the sweet proteins (Benincasa hispida thaumatin-like proteins) of the melon class.

Disclosure of Invention

In view of the problems existing in the prior art, the invention discloses a white gourd sweet protein gene, white gourd sweet protein, engineering bacteria containing the gene and application thereof in antifungal aspect.

The technical scheme for solving the technical problems is as follows:

The invention provides a white gourd sweet protein, which comprises an amino acid sequence shown as SEQ ID NO. 1. The protein provided by the invention can inhibit the growth of plant pathogenic fungi fusarium oxysporum and has good antifungal activity. The gene can be used for producing protein preparation, so that the protein preparation can be widely applied to biological control of agricultural plant pathogenic fungi, and considerable economic benefit can be obtained while the actual problem is solved.

The invention provides a wax gourd sweet protein gene, which codes the wax gourd sweet protein.

The sweet protein gene of the wax gourd can comprise a nucleotide sequence shown as SEQ ID NO. 2. The full length of the gene (from the start codon to the stop codon) is 681bp, G+C% is 53.3%, 226 amino acids are encoded, and the theoretical molecular weight of the protein is 25.1kDa.

The sweet protein gene of the wax gourd can also comprise an optimized sweet protein gene sequence of the wax gourd, and the optimized sweet protein gene of the wax gourd comprises a nucleotide sequence shown as SEQ ID NO. 3.

The sweet protein gene of the waxgourd is discovered for the first time, and the sweet protein can be obtained by encoding the sweet protein gene.

The invention provides a vector comprising the sweet protein gene of the waxgourd. The vector can be a cloning vector or an expression vector. For example, the recombinant vector may be used, and preferably, the above-mentioned wax gourd sweet protein gene may be cloned into pPICZαA.

The present invention provides a microorganism comprising the above-mentioned wax gourd-like sweet protein gene, which may be carried in various ways, including, but not limited to, in a plasmid, on a genome, etc. For example, a recombinant microorganism containing the above recombinant vector; preferably, pichia pastoris (Pichia pastoris X-33) is used as host bacteria. The Pichia pastoris engineering strain constructed by the gene can efficiently express the sweet proteins of the cucurbitaceous plants.

The invention provides application of one or more of the sweet proteins of the waxgourd, the sweet protein genes of the waxgourd, the carrier and the microorganism in antibiosis. For example, can be used against plant pathogens; can be applied to inhibiting the growth of plant pathogenic fungi; can be used for preparing protein preparation.

The white gourd sweet protein can be used as an antibacterial protein in the aspect of agricultural plant pathogenic fungi resistance.

In the above application, the plant pathogenic bacteria may be Fusarium oxysporum. The vector can be obtained by cloning the sweet protein gene of the cucumis melo into pPICZ alpha A. The microorganism can take Pichia pastoris X-33 as host bacteria. The sweet protein gene of the wax gourd can comprise a nucleotide sequence shown as SEQ ID NO. 2 and/or an optimized sequence. The optimized sequence may include the nucleotide sequence shown in SEQ ID NO. 3.

The expression level of the white gourd sweet protein gene provided by the invention is up-regulated in white gourd root tissues induced by fusarium wilt bacteria; the white gourd sweet protein provided by the invention has the activity of inhibiting the sporulation of fusarium wilt.

The invention also provides a method for resisting fusarium oxysporum, which comprises the following steps: the sweet proteins of the wax gourd class are adopted to resist fusarium oxysporum.

The preparation method of the white gourd sweet protein can comprise the following steps: constructing a recombinant vector containing the sweet protein genes of the waxgourd, constructing a recombinant strain for expressing the sweet protein genes of the waxgourd by taking pichia pastoris as host bacteria, and obtaining the sweet protein of the waxgourd by using methanol for induction expression.

The invention provides a theoretical basis for the research and development of key technology for preventing and controlling plant fungal diseases by screening antifungal plant genes, protein coded by the antifungal plant genes and disclosing the action mechanism of the antifungal protein, and provides technical guarantee for realizing green high-quality production of crops by applying the antifungal plant genes to biological prevention and control.

Drawings

FIG. 1 is a schematic diagram of the cloning of a sweet protein gene of white gourd class.

FIG. 2 is a diagram showing an experimental scheme of high-efficiency expression of the sweet protein gene of white gourd in P.pastoris X-33 (pPICZ. Alpha.A).

FIG. 3 is the results of colony PCR validation; wherein: DL5000 nucleic acid Marker;1-8 randomly 8 clones were picked from the plates.

FIG. 4 shows the results of detecting the expression of a target protein by using a dye; the upper graph shows the result of examination, in the upper graph, M is Marker,1-7: protein of interest (clone 1 to clone 7), 8: a positive control; the lower graph shows experimental results of WB, in the lower graph, 1-7: the target proteins (clone 1 to clone 7) were each in that order.

FIG. 5 shows experimental results of protein amplification expression detection, wherein M: marker,1, target protein, 2: positive control.

FIG. 6 shows the results of purified protein assays, wherein M: marker,1: bhTLP22 effluent, 2: washing impurity liquid, 3: purifying the protein.

FIG. 7 is a standard curve of protein concentration determination.

FIG. 8 Fusarium oxysporum induced expression of BhTLP.sup.1 and BhTLP in white gourd root tissue.

Fig. 9 is an experimental result for the effect of recombinant sweet proteins of the wax gourd class on fusarium oxysporum, wherein: control (CK): an execution buffer; treatment group (BhTLP-treated): adding recombinant sweet wax gourd protein BhTLP; a-d are plate photographs of Fusarium oxysporum growth (8 days of culture); e. f effects on Fusarium oxysporum (8 days of culture) cells; g is a graph of Fusarium oxysporum diameter, and the abscissa dpi is a shorthand for the days post-inoculation.

Detailed Description

The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.

The invention uses the transcriptome data of the variety of white gourd and refers to the genome sequence information of white gourd, and the sequence of the white gourd sweet protein (Benincasa hispida thaumatin-like protein) gene is successfully obtained through PCR amplification. The full length of the gene (from the start codon to the stop codon) is 681bp, G+C% is 53.3%, and 226 amino acids are encoded.

The amino acid sequence of the sweet protein of the wax gourd class is shown as SEQ ID NO. 1:

MGISNICFLFFLALLSFYSQTHAATFQVRNNCPFTVWAAAVPGGGRRLNRNDVWTFNVNPGTVAARIWPRTNCNFDGSGRGRCQTGDCGGLLQCQAYGTPPNTLAEYALNQFNNLDFFDISLVDGFNVPMEFSPTSGGCTRGIRCTADINGQCPNELRAPGGCNNPCTVFGGDRYCCTAPNSSCGPTDYSRFFKNRCPDAYSYPKDDATSTFTCPGGTHYRVVFCP

the full-length nucleotide sequence of the sweet protein gene of the wax gourd is shown as SEQ ID NO. 2:

ATGGGTATCTCTAATATTTGTTTTCTCTTCTTTCTTGCTCTTCTTTCTTTCTATTCCCAAACCCATGCAGCTACTTTTCAAGTCCGAAACAACTGCCCCTTCACTGTTTGGGCTGCTGCAGTACCCGGCGGTGGACGACGACTTAATCGAAACGATGTTTGGACATTTAACGTGAATCCGGGCACCGTTGCTGCTCGTATTTGGCCTCGAACTAACTGCAACTTCGACGGTTCTGGCCGGGGTAGATGCCAGACTGGCGACTGTGGTGGTCTCCTCCAGTGCCAAGCTTACGGCACTCCGCCGAACACCCTTGCCGAATACGCGTTAAATCAGTTCAATAACTTGGACTTCTTCGATATCTCTCTCGTCGATGGATTTAACGTTCCGATGGAGTTCAGCCCGACTTCTGGGGGGTGCACTCGTGGCATCAGGTGCACCGCGGACATCAACGGACAGTGCCCGAACGAGCTTAGGGCTCCTGGAGGGTGCAACAACCCGTGCACCGTGTTCGGGGGTGATCGATACTGCTGCACCGCCCCCAACAGCAGCTGTGGCCCGACGGATTACTCTAGGTTCTTCAAGAATCGGTGCCCAGATGCATACAGTTACCCAAAAGACGATGCAACCAGCACATTCACTTGCCCCGGGGGAACCCACTACAGGGTTGTCTTCTGTCCTTGA

When cloning the white gourd sweet protein gene, pichia pastoris can be used as an engineering strain, as shown in figure 1, and the method comprises the following steps: and (3) removing signal peptide and codon from the sweet protein genes of the white gourd, synthesizing the optimized target genes to pPICZ alpha A by a gene synthesis technology, wherein the target genes are close to the vector alpha-factor, and the C terminal band is 6 XHis, so as to obtain the target vector. Linearizing and purifying the target vector, recovering, preparing yeast P.pastoris X-33 electrotransformation competence, electrotransformation, screening the transformant, and utilizing methanol to induce expression. By the method, the white gourd sweet protein is efficiently expressed by taking pichia pastoris as an engineering strain, and the induced expression quantity can be 148ug/ml.

The engineering strain constructed by the gene can efficiently express the sweet protein of the winter melon, when the wild WT of the fusarium oxysporum is taken as a target, after the wild WT is cultured on a culture medium added with the recombinant sweet protein BhTLP of the winter melon, the growth of the fusarium oxysporum is obviously inhibited, and compared with a control group, the fusarium oxysporum in a treatment group dies more seriously through microscopic observation. The protein can inhibit the growth of plant pathogenic fungi Fusarium oxysporum and has good antifungal activity. The protein preparation produced by the gene can be used for biological control of agricultural plant pathogenic fungi, and can obtain considerable economic benefit while solving the practical problem.

In the invention, unless specified, the experimental methods used are all conventional experimental methods in the field; the materials, reagents and apparatus used are all materials, reagents and apparatus conventional in the art and are commercially available or can be prepared by methods conventional in the art.

PPICZ alpha A was purchased from Nanjing Rayleigh sources biotechnology Co., ltd; host bacteria p.pastoris X-33 was purchased from nanjing raysource biotechnology limited; fusarium oxysporum is purchased from the Chinese strain resource library collection center.

The buffer A is prepared according to the following proportion: 20ml YPD+2ml 2M 4-hydroxyethylpiperazine ethanesulfonic acid HEPES (pH=8.0, filter sterilization) +0.5ml 1M dithiothreitol DTT (filter sterilization).

The Lysis buffer formula comprises: formulated with water, 50mM NaH ₂PO₄, 300mM NaCl,10mM imidazole,pH =8.0, and sterilized by filtration.

The Wash buffer formula comprises: formulated with water, 50mM NaH ₂PO₄, 300mM NaCl,20mM imidazole,pH =8.0, and sterilized by filtration.

The formulation of the solution buffer comprises: formulated with water, 50mM NaH ₂PO₄, 300mM NaCl,250mM imidazole,pH =8.0, and sterilized by filtration.

YPD: 1% of yeast extract, 2% of peptone and 2% of glucose, and if a solid culture medium is prepared, 2% of agar powder is added, wherein the mass percentages of the above components are all.

YPG medium: 1% of yeast extract, 2% of peptone, 3% of ethanol and 3% of glycerol, wherein if a solid culture medium is prepared, 2% of agar powder is added, and the mass percentages are all the above.

The PDA culture medium preparation method can be prepared according to the following proportion: cutting peeled potato into pieces, adding 800mL of water, boiling for 20 minutes, filtering with gauze, adding 20g of glucose and 15g of agar powder, fixing the volume to 1L, and sterilizing at 121 ℃ for 20 minutes by high-pressure steam; after sterilization, the medium with the temperature reduced to about 50 ℃ is poured into a sterile culture dish to prepare the PDA plate.

The liquid Chlamydia medium is prepared according to the following proportion: 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate (MgSO ₄·7H₂ O), 0.5g of potassium chloride, 0.01g of ferrous sulfate, 30g of sucrose and 1000mL of distilled water.

Sodium nitrate was purchased from national pharmaceutical group chemical reagent limited, cat: 10019928; dipotassium hydrogen phosphate is purchased from national pharmaceutical group chemical reagent company, cat: HX10025; mgSO ₄·7H₂ O was purchased from Guogu chemical Co., ltd., product number: XW221890881; potassium chloride was purchased from national pharmaceutical groups chemical reagent Co., ltd., product number: 10016308; ferrous sulfate was purchased from Shanghai source leaf Biotechnology Co., ltd., cat: t20691; sucrose was purchased from national pharmaceutical group chemical reagent company, cat No.: 10021487;10 Xbuffer was purchased from Siemens; sacI was purchased from sameid, cat No.: ER1131; the nucleic acid precipitation aid is purchased from Beijing Soy Bao technology Co., ltd., product number: SA1020; BMMY medium was purchased from the Probiotechnological engineering (Shanghai) Co., ltd. (abbreviated: protect), cat: b540131-0500; protein loading buffer available from Beijing full gold Biotechnology Co., ltd., product number: DL101-02; ECL is purchased from the manufacturer, cat No.: c510045-0100; gravity columns were purchased from beijing solebao technologies, inc., product number: YA2450; ni-NTA beads were purchased from Beijing orchid Boli Biotechnology Co., ltd., product number: n30210; the polysaccharide polyphenol plant total RNA extraction kit is purchased from Tiangen Biochemical technology (Beijing) limited company, product number: DP441; seqHunt FIRST STRANG CDNA SYNTHESIS KIT is available from the entropy theory (Shenzhen biotechnology Co., ltd; the Bradford method protein concentration assay kit was purchased from the biotechnology company, huawa, cat No.: HYJYKY5975;2x U+SYBR qPCR Master Mix available from entropy theory (Beijing) Biotechnology Inc., cat# BC05-100.

In each example, the sweet protein gene of the wax gourd class is simply referred to as BhTLP gene for convenience of description.

The following is presented by way of specific examples.

Example 1

As shown in fig. 2, the high-efficiency expression of the sweet protein gene of the cucumis melo in pichia pastoris comprises the following steps: firstly, constructing a recombinant vector comprising an optimized BhTLP gene; secondly, linearizing the target carrier; finally, the transformants were electrotransformed into Pichia pastoris X-33 and screened.

1.1 Construction of recombinant vector pPICZ alpha A-BhTLP22

Reference is made to white gourd Benincasa hispida thaumatin-like protein (LOC 120067974), which is derived from NCBI database search.

The CDS region of the gene is subjected to codon optimization, and sequence codons can be better expressed in yeast through optimization. The optimized gene sequence is synthesized by Shanghai qing biological technology Co-Ltd by adopting a chemical method, the optimized gene sequence is directly synthesized to pPICZ alpha A, the target gene is next to the alpha-factor of the vector pPICZ alpha A, and the C terminal band is 6 XHis. And (3) verifying by using an XhoI/SmaI enzyme digestion method to obtain the correct recombinant vector pPICZ alpha A-BhTLP.

The optimized gene nucleotide sequence is shown as SEQ ID NO. 3:

GCTACTTTCCAAGTTAGAAACAACTGTCCATTCACTGTTTGGGCTGCTGCTGTTCCAGGTGGTGGTAGAAGATTGAACAGAAACGATGTTTGGACTTTCAACGTTAACCCAGGTACTGTTGCTGCTAGAATCTGGCCAAGAACTAACTGTAACTTCGATGGTTCTGGTAGAGGTAGATGTCAAACTGGTGATTGTGGTGGTTTGTTGCAATGTCAAGCTTACGGTACTCCACCAAACACTTTGGCTGAATACGCTTTGAACCAATTCAACAACTTGGATTTCTTCGATATCTCTTTGGTTGATGGTTTCAACGTTCCAATGGAATTCTCTCCAACTTCTGGTGGTTGTACTAGAGGTATCAGATGTACTGCTGATATCAACGGTCAATGTCCAAACGAATTGAGAGCCCCAGGTGGTTGTAACAACCCATGTACTGTTTTCGGTGGTGATAGATACTGTTGTACTGCTCCAAACTCTTCTTGTGGTCCAACTGATTACTCTAGATTCTTCAAGAACAGATGTCCAGATGCTTACTCTTACCCAAAGGATGATGCTACTTCTACTTTCACTTGTCCAGGTGGTACTCATTACAGAGTTGTTTTCTGTCCA

1.2 mesh Carrier linearization

The recombinant plasmid (pPICZ. Alpha. A-BhTLP) constructed above was linearized with the restriction enzyme SacI, comprising the steps of:

Firstly, preparing a carrier enzyme digestion system, wherein the enzyme digestion system comprises: the plasmid (5-10 ug) was converted to a volume of 6ul, 10 Xbuffer 5ul, sacI 1ul, and the 50ul system was filled with ddH ₂ O.

Secondly, placing the enzyme digestion system at 37 ℃ for reaction for 8 hours, recovering enzyme digestion products, and inactivating the reaction system at 65 ℃ for 20 minutes after the enzyme digestion is detected to be successful.

1.3 Purification recovery of linearized Carriers by ethanol precipitation

The method adopts an ethanol precipitation method to purify and recycle the linearization carrier and comprises the following steps:

(1) Preparing a carrier purification system: 50ul of inactivated cleavage product (prepared in example 1.2), 10ul of nucleic acid precipitation aid, 6ul of 3M NaAc (pH=5.2), 165ul of absolute ethanol;

(2) Standing the above system at-20deg.C for more than 35 min; centrifuging at 12000rpm at 4deg.C for 15min, discarding supernatant, at which time white precipitate on the wall can be observed; adding 400ul of pre-cooled 80% ethanol, re-suspending, precipitating, centrifuging at 12000rpm for 10min at 4deg.C, discarding supernatant, and uncovering for drying; the precipitate was dissolved by adding 10ul of ddH ₂ O.

1.4 Preparation of Yeast electric transfer competence

The preparation method of the yeast electrotransformation competence comprises the following steps: 5ml YPD was added to a 50ml centrifuge tube, and P.pastoris X-33 strain was inoculated and cultured overnight at 30 ℃; transferring 50ul of bacterial liquid to a 250ml conical flask of 50ml YPD at about 1 pm in the next day, and culturing overnight until OD ₆₀₀ = 1.3-1.5; centrifuge at 4000rpm for 5min at 4℃and resuspend with 10ml buffer A, water bath at 30℃for 15min and add pre-chilled sterile water to 50ml. Centrifuging at 4000rpm at 4 ℃ for 5min, re-suspending with 50ml of pre-cooled heavy suspension, wherein the heavy suspension is prepared according to the following proportion: 50ml of sterile water +0.3ml 2M HEPES,pH =8.0; centrifuge at 4000rpm for 5min at 4℃and re-suspend with 4ml of pre-chilled 1M sorbitol (sorbitol); centrifugation was performed at 4000rpm for 5min at 4℃and 100ul of pre-chilled 1M sorbitol (in which case the bacterial suspension was viscous) and 80 ul/tube was dispensed and placed on ice.

1.5 Electric transformation of Pichia pastoris with linearization vector

Electrotransformation of the recovered purified linearized vector into pichia pastoris, comprising the steps of: 80ul P.pastoris X-33 competent cells (prepared by the method of example 1.4) were taken, 6ug of linearized pPicZ αA-BhTLP22 (prepared by the method of example 1.3) were added, mixed well and transferred to a pre-chilled 0.2cm electric shock cup; placing on ice for 5min; setting electric shock parameters (1.5 kV, 25uF, 200Ω) for Pichia pastoris, and electric shock; immediately adding 2ml of precooling liquid, wherein the precooling liquid is prepared according to the following proportion: 10ml 1M sorbitol+100ul 2M HEPES,pH = 8.0; transfer to a 2ml sterile centrifuge tube; standing and incubating for 1-2h at 30 ℃; after dilution 5-fold, 10-fold and 100-fold, the cells were plated on YPD plates (15 cm in diameter) containing 100mg/l bleomycin (Zeocin) at 300 ul/plate, and cultured at 30℃until clones were developed.

1.6 Selection of transformants

Screening transformants, comprising the steps of: randomly selecting 8 clones from the plate, and performing colony PCR;

The sequence of the forward primer adopted in the colony PCR process is shown as SEQ ID NO. 4: CAAAAAAAGAGATCTTTAATACGACTCACTATAGGGCGAGCGCCGCCATG.

The sequence of the reverse primer adopted in the colony PCR process is shown as SEQ ID NO. 5: CACGATGCACAGTTGAAGTGAACTTGCGGGGTTTTTCAGTATCTACGATT.

Colony PCR used was a rapid amplification yeast cloning kit (product number RY 8001) developed independently by Nanjing Resource Biotechnology Co., ltd; the colony PCR products are subjected to electrophoresis, the experimental result is shown in figure 3, and the size of the electrophoresis strip is consistent with that expected; 2 of them are selected for sequencing, and after the comparison is correct, the sequence is reserved for later use. Pichia pastoris recombinant strains containing BhTLP are obtained by the method.

Example 2 efficient expression of a Benincasa sweet protein Gene in Pichia pastoris

2.1 Transformant expression miniprep

A transformant expression panel comprising the steps of: selecting 7 clones (prepared by the method of example 1 and named clone 1 to clone 7 respectively) of the recombinant Pichia pastoris strain containing BhTLP which are verified to be correct, respectively inoculating 50ml conical flasks, filling 5ml YPG culture medium in each conical flask, and culturing at 30 ℃ for 1-2 days at 220rpm until bacterial liquid is saturated; transferring into a 50ml centrifuge tube, centrifuging at 4000rpm for 5min, and discarding the supernatant; the cells were resuspended in 5ml BMMY medium, transferred to a new sterile 50ml Erlenmeyer flask, added with methanol to a final concentration of 0.75% (volume percent) and incubated at 28℃at 220rpm for 6d; methanol is volatile, and is added every 24 hours, so that the final concentration of the methanol is 0.75 percent (volume percent); bacterial liquid was collected at 6d am, centrifuged at 4000rpm for 5min, and supernatant was collected for examination and WB identification to investigate expression.

The examination and WB identification method comprises the following steps: taking 40ul of protein solution (i.e. the supernatant prepared by the method), adding a proper amount of protein loading buffer, boiling at 100 ℃ for 5-10min; centrifuging at 12000rpm for 2min, respectively sucking 20ul of the solution, performing protein electrophoresis, separating out the laminated gel at 80V, and then finishing electrophoresis at 100V; one of the two is used for coomassie brilliant blue dyeing, and the other is used for film transfer, 100V and 90min; washing PVDF film with TBST for 1 time after finishing film transfer, 10min, and pouring out TBST; blocking with TBST containing 5% skimmed milk powder, and blocking on a shaker at 50rpm at room temperature for 1 hr; diluting the primary antibody with a blocking solution containing 5% skimmed milk powder, and hybridizing the membrane on a low-speed shaker at room temperature for 4h: TBST washes the membrane for 3 times, each time for 10min; diluting the secondary antibody with a sealing solution containing 5% skimmed milk powder, and hybridizing the membrane on a low-speed shaking table at room temperature for 45min; TBST washes the membrane for 3 times, each time for 10min; after ECL exposure, the films were scanned by a machine.

The experimental results are shown in FIG. 4, in which the upper graph is the experimental results of the test of clone 1 to clone 7, and it is hypothesized that no band was seen, possibly because of low test sensitivity and no specificity, but that no band was seen does not represent no band of interest. Therefore, further experiments with WB were used to verify whether clone 1 through clone 7 expressed the protein of interest, and it can be seen from the lower panel of FIG. 4 that clone 2, clone 4, clone 6 and clone 7 were successfully expressed, presumably due to differences exhibited after transformation of yeast with the vector or experimental manipulations, etc., resulting in unsuccessful expression of some clones. Of the clones that can be expressed, clone 7 is most desirably expressed, and thus clone 7 is selected for subsequent testing.

2.2 Optimal expanded expression of transformants

The optimal transformant (clone 7 in example 2.1) was expanded for expression, comprising the following steps: 50ml YPG medium is added into a 250ml conical flask, the optimal expression transformant (clone 7 in example 2.1) is inoculated, and the culture is carried out at 30 ℃ and 220rpm for 1-2 days until bacterial liquid is saturated; transferring into a 50m l centrifuge tube, centrifuging at 4000rpm for 5min, and discarding the supernatant; the cells were resuspended in 50ml BMMY medium, transferred to a new sterile 250ml Erlenmeyer flask, added with methanol to a final concentration of 0.5% (volume percent) and incubated at 28℃at 220rpm for 6d; adding methanol every 24h to make the final concentration of methanol be 0.75% (volume percent); bacterial liquid was collected at day 6, centrifuged at 4000rpm for 5min, and the supernatant was collected for expression identification.

The specific method for expression identification by using examination and WB identification is the same as in example 2.1.

As shown in FIG. 5, it can be seen from FIG. 5 that the expanded expression of positive clone 7 was successful.

2.3 Protein purification

The sample for expression identification is subjected to protein purification, and the method comprises the following steps: 5ml of Ni-NTA beads are filled in a 30ml gravity column; the column was equilibrated with 5ml Lysis buffer and repeated 3 times; the 50m l protein solution (i.e., bhTLP effluent) was passed through the column twice; adding 10ml Wash buffer for washing impurities, repeating for 5 times, and reserving the impurity washing liquid for subsequent SDS-PAGE electrophoresis; adding 5ml Elution buffer for eluting to obtain purified protein.

SDS-PAGE of purified recombinant sweet proteins of the wax gourd class is carried out, and the result is shown in FIG. 6, wherein the protein sample is successfully purified without other bands.

The concentration of the purified protein is detected by using a protein concentration determination kit according to the Bradford method, and the method comprises the following steps of: diluting the BSA standard to 1mg/ml as required; and (3) making a standard curve, and calculating the concentration of the sample according to the standard curve.

The standard curve is prepared by the following steps: preparing BSA standard substance solutions with different concentrations, wherein the BSA concentrations are respectively 0ug/ml, 25ug/ml, 50ug/ml, 75ug/ml and 100ug/ml; 20ul of the solution is respectively added with 180ul of coomassie brilliant blue G250 dye, and the mixture is uniformly mixed, and the absorbance at 595nm is measured by an enzyme-labeled instrument. The standard curve equation is plotted with absorbance values on the abscissa and BSA concentration (ug/ml) on the ordinate, as shown in fig. 7.

Taking 20ul of purified protein sample to be detected, adding 180ul of coomassie brilliant blue G250 dye, uniformly mixing, measuring the absorbance value of 595nm by using an enzyme-labeled instrument, calculating the concentration of the protein according to the detected absorbance value and a standard curve equation, and detecting that the concentration of the protein in the sample is about 148.08 mug/ml.

EXAMPLE 3 Induction test of white gourd root by fusarium wilt

The experimental method comprises the following steps:

(1) Flat plate culture of fusarium wilt bacteria: fusarium oxysporum was inoculated onto PDA plates, cultured at 26℃in the dark for 5 days, and then used for liquid culture.

(2) Liquid culture of wilt: fusarium oxysporum on a PDA culture medium is picked by a sterilized toothpick, inoculated in a liquid Soxhlet culture medium together with the PDA culture medium, cultured for 3 days at 26 ℃ and 200rpm, and then the Fusarium oxysporum spores are collected by a centrifugal method, and resuspended in sterile water.

(3) Inoculation experiment: the method comprises the steps of adopting water culture to achieve two-liquid and one-heart white gourd seedlings, adding the prepared bacterial liquid into a water culture box until the concentration is 1X10 ⁶ pieces/ml, respectively taking root tissues treated for 0h, 3h, 12h and 48h, treating the root tissues with liquid nitrogen, and preserving the root tissues at the temperature of minus 80 ℃.

(4) Extracting RNA of white gourd root tissues: extracting RNA according to the operating instructions of the polysaccharide polyphenol plant total RNA extraction kit.

(5) First strand cDNA was synthesized using SeqHunt FIRST STRANG CDNA SYNTHESIS KIT, comprising the steps of:

① Template RNA was taken and primers were added, and the system (total volume 14. Mu.l) included: total RNA 1. Mu.g, random Primer (Random Primers) 1. Mu.l, oligo (dT) ₁₅ Primer 1. Mu.l, and nucleic-FREE WATER were made up to 14. Mu.l. The mixture of template RNA and primer is pre-denatured at 65 ℃ for 5min, and the mixture is taken out and placed on ice after the completion of the pre-denaturation, so that a mixed sample is obtained. The random Primer (Random Primers) and Oligo (dT) ₁₅ Primer were SeqHunt FIRST STRANG CDNA SYNTHESIS KIT self-contained.

② The reaction system was prepared and 10. Mu.l, 2. Mu.l, 5X Reaction Buffer 4. Mu.l of the mixed sample of step ①, 10 Xenzyme Mix, were added to each sample tube. Gently beating and mixing by a pipette. The first strand cDNA synthesis reaction was performed under the following conditions: 25 ℃ for 5min;50 ℃ for 30min;85 ℃ for 2min. cDNA was obtained.

(6) Real-time quantitative reverse transcription PCR (quantitative reverse transcriptional PCR, qRT-PCR):

preparing a reaction system (total volume of 20 μl) by taking the cDNA obtained in the step (5) as a template for real-time quantitative PCR: 2x U ⁺ SYBR QPCR MASTER Mix 10. Mu.l, forward primer 0.4. Mu.l, reverse primer 0.4. Mu.l, template cDNA 2. Mu.l, nuclear-FREE WATER make up to 20. Mu.l.

The forward primer for detection BhTLP1 is GGCGTTTGCAAATCCGAACA (SEQ ID NO: 6); the reverse primer for detection BhTLP1 was CTGAAATGTCAGCCGCTTGC (SEQ ID NO: 7).

The forward primer for detection BhTLP22 is CGACTGTGGTGGTCTCCTCC (SEQ ID NO: 8); the reverse primer for detection BhTLP was AGTCGGGCTGAACTCCATCG (SEQ ID NO: 9).

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

Cycling procedure for real-time quantitative PCR: ①95℃,5min;②95℃,15sec;③60℃,40sec;④Go to②,40cycles;⑤ Melting curve: qPCR instrument was self-contained.

The experimental results are shown in FIG. 8, and it can be seen from FIG. 8 that under the induction of Fusarium oxysporum, bhTLP22 significantly up-regulates the expression at 3h, 12h and 48h compared with BhTLP 1.

Example 4 validation of the effect of BhTLP protein on Fusarium oxysporum plate colonies

The culture medium supplemented with BhTLP protein was used as a treatment group, and the plate without BhTLP protein was used as a control group.

In the treatment group, bhTLP protein solution (prepared by the method of example 2.3) was added to PDA medium to a final concentration of BhTLP protein of 0.2mM.

In the control group (CK), the BhTLP protein solution was replaced with an equivalent amount of the Elution buffer.

Fusarium oxysporum was inoculated onto PDA medium plates, the inoculated plates were cultured at 26℃in the dark, and the diameters of colonies cultured for 2 days, 4 days, 6 days, and 8 days were measured.

As shown in FIG. 9, compared with the CK of the control group, the fusarium oxysporum colony on the plate added with BhTLP protein has smaller diameter and slower growth, so that the BhTLP protein has obvious inhibition effect on the fusarium oxysporum growth. BhTLP22 protein can be used for preparing a preparation for preventing and treating fusarium oxysporum, and can be used for preventing and treating diseases caused by fusarium oxysporum.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

Claims

1. The application of the wax gourd sweet protein in resisting fusarium oxysporum is characterized in that the amino acid sequence of the wax gourd sweet protein is shown as SEQ ID NO. 1.

2. The application of the sweet melon protein gene in resisting fusarium oxysporum is characterized in that the sweet melon protein gene codes sweet melon protein, and the amino acid sequence of the sweet melon protein is shown as SEQ ID NO. 1.

3. The use according to claim 2, wherein the sweet melon protein gene comprises the nucleotide sequence shown in SEQ ID No. 2 or an optimized sequence.

4. The application of the vector comprising the wax gourd sweet protein gene in resisting fusarium oxysporum is characterized in that the wax gourd sweet protein gene codes the wax gourd sweet protein, and the amino acid sequence of the wax gourd sweet protein is shown as SEQ ID NO. 1.

5. The use according to claim 4, wherein the vector is obtained by cloning a sweet protein gene of the family Benincase into pPICZ alpha A.

6. The application of the microorganism comprising the wax gourd sweet protein gene in resisting fusarium oxysporum is characterized in that the wax gourd sweet protein gene codes the wax gourd sweet protein, and the amino acid sequence of the wax gourd sweet protein is shown as SEQ ID NO. 1.

7. The use according to claim 6, wherein the microorganism is pichia pastoris X-33 as host bacteria.