CN108441458B

CN108441458B - Genetically engineered bacillus amyloliquefaciens capable of effectively preventing and treating gray mold as well as preparation method and application thereof

Info

Publication number: CN108441458B
Application number: CN201711172205.8A
Authority: CN
Inventors: 王傲雪; 王旭东; 陈秀玲; 张淑梅; 张瑶; 刘佳音
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2017-11-22
Filing date: 2017-11-22
Publication date: 2021-05-25
Anticipated expiration: 2037-11-22
Also published as: CN108441458A

Abstract

Genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold as well as a preparation method and application thereof belong to the field of molecular biology. The invention amplifies the chitinase gene ech42 of the gliocladium roseum through gene engineering, connects with a degradation expression vector pHT43, constructs a recombinant expression vector pHT43/ech42, and introduces the recombinant expression vector pHT43/ech42 into the bacillus amyloliquefaciens through setting electric shock transformation, wherein the electric shock voltage is 12.5kv/cm, 15kv/cm or 17.5 kv/cm. The enzyme activity of pHT43-ech42/BA strain at the 8 th hour in the chitinase activity detection is improved by about 54.3 percent compared with the enzyme activity of BA of a wild strain, the disease prevention effect is still about 28.9 percent after 20 days of fungus spraying in the aspect of gray mold prevention, and the chitinase activity detection has a lasting biological prevention effect.

Description

Genetically engineered bacillus amyloliquefaciens capable of effectively preventing and treating gray mold as well as preparation method and application thereof

Technical Field

The invention belongs to the field of molecular biology; in particular to genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold as well as a preparation method and application thereof.

Background

Fungal plant diseases are main factors for restricting the high yield of plants, the epidemic of the fungal diseases causes extremely serious yield reduction of crops, and the crops such as tomatoes are often attacked by the fungal diseases such as botrytis cinerea, phyllothyrium, root rot, early blight and the like, so that the yield reduction of tomatoes is large. At present, the main prevention and treatment means for preventing and treating the gray mold is chemical prevention and treatment, which can pollute the environment, and the problems of pesticide residue and the like also harm the health of human bodies; the method for preventing and treating the gray mold by the breeding for disease resistance is expected to achieve thorough prevention and treatment, but no antigen material for the gray mold is found at present, so that the prevention and treatment of the gray mold by the breeding for disease resistance is not feasible; at present, the method for preventing and treating fungal diseases by biological control and using some environment-friendly and harmless microorganisms is an optimal solution, can not only fill up the defects of chemical control, but also ensure the cleanness and sanitation of crops.

The gliocladium roseum can prevent and treat the harm of pathogenic bacteria by various disease-resistant mechanisms like other biocontrol bacteria, and can be mainly prevented and treated in 5 aspects: (1) competitive action, (2) bacteriolysis action, (3) antibiotic action, (4) heavy parasitic action, and (5) plant disease resistance induction. The bacteriolysis mainly produces chitinase, namely chitin hydrolase, so that the cell wall of pathogenic fungi is dissolved, and the plasma membrane of the pathogenic fungi is cracked, thereby inhibiting the growth and the reproduction of the pathogenic fungi.

Bacillus amyloliquefaciens is widely distributed in nature, and can control fungal diseases through various bacteriostatic mechanisms, mainly comprising the following components: (1) generating bacteriostatic substances. The bacillus amyloliquefaciens mainly acts by producing substances with the effect of inhibiting the growth of pathogenic bacteria hyphae, and the antibacterial substances mainly comprise antibacterial proteins, lipopeptide antibiotics and the like. (2) Disease resistance was induced. The antagonistic microbe is used to treat fruit, which can result in morphological change of host and physiological and biochemical change of host, and further result in the change of expression level of host plant protein group and disease-resistant related gene. (3) Compete with pathogenic bacteria for living space and nutrients. (4) Can directly act on pathogenic bacteria. In addition to producing secondary metabolites such as antibiotics, antagonistic bacteria also produce a series of extracellular hydrolases, such as chitinase, which play an important role in the inhibition of pathogenic bacteria.

At present, many commercial products are sold in the market, and biological pesticides prepared by antagonistic microorganisms of pathogenic bacteria are used for preventing and treating plant diseases, such as bacillus subtilis, bacillus licheniformis, pseudomonas fluorescens, bacillus cereus, trichoderma, bacillus thuringiensis, bacillus amyloliquefaciens and the like, and the biological control bacteria have good effects in the process of preventing and treating the plant diseases. So that it is possible to control pathogenic bacteria by biocontrol bacteria.

Disclosure of Invention

The invention aims to provide genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold as well as a preparation method and application thereof.

The invention is realized by the following technical scheme:

a genetically engineered Bacillus amyloliquefaciens capable of effectively preventing and treating gray mold is classified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), wherein a chitinase gene of Gliocladium roseum is transferred into the Bacillus amyloliquefaciens, and the Bacillus amyloliquefaciens is preserved in a China center for microbiological culture Collection center (CGMCC) in 29 th 6 th 2017, and has a preservation number of CGMCC No.14366 and a preservation address of No. 3 North Chen Lu No.1 of the sunward area in Beijing.

The preparation method of the genetic engineering bacillus amyloliquefaciens for effectively preventing and treating the gray mold comprises the following steps of:

step A, amplifying chitinase gene ech42 of Gliocladium roseum through genetic engineering, connecting an expression vector pHT43, and constructing a recombinant expression vector pHT43/ech 42;

and step B, introducing the recombinant expression vector pHT43/ech42 in the step 1 into the bacillus amyloliquefaciens by setting electric shock transformation, wherein the electric shock voltage is 12.5kv/cm or 15kv/cm or 17.5 kv/cm.

step a1, obtaining c.rosea chitinase gene ech42 by PCR cloning;

step A2, connecting the chitinase gene ech42 obtained in the step A1 with a T vector;

step A3, connecting the chitinase gene ech42 obtained in step 2 with an expression vector pHT 43.

The preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, disclosed by the invention, comprises the following steps of finding out a gene sequence of a C.rosea chitinase ech42 gene (DQ523687) through GenBank in a step A1, detecting the signal peptide sequence by using signal peptide detection software, designing a primer by using primmer5 software, wherein a cloned gene does not contain a restriction enzyme site introduced into the primer:

primer ech 42E: 5'-TATCTAGACGTGCCACTCCTCGTATGGAGG-3', the restriction enzyme cutting site Xba I;

ech42, 42B: 5'-CCCCCCGGGGGAGAGGCTGTTCTTGAT-3', the restriction enzyme cutting site Sma I;

the mixture of the PCR reactions was: template 1. mu.l, 10. mu.M upstream primer ech42E 1. mu.l, 10. mu.M downstream primer ech42B 1. mu.l, 10 × EasyTaq buffer 5. mu.l, 2.5mM dNTPs 4. mu.l, EasyTaq DNA polymerase 0.5. mu.l, ultrapure water 37.5. mu.l;

the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 0.5min, annealing at 68 deg.C for 0.5min, extension at 72 deg.C for 1min, and 35 cycles; finally, after extension at 72 ℃ for 5min, the cells were stored at 4 ℃.

The preparation method of the genetic engineering bacillus amyloliquefaciens for effectively preventing and treating gray mold comprises the following steps of A2, recovering and purifying PCR amplification products by using a reference Kit StarPrep Gel Extraction Kit; connecting the recovered target gene ech42 with a T vector, wherein the reaction system is 1 mul of pMD18-T vector, 1 mul of target gene ech424 mul and 5 mul of solution I; and (3) carrying out single enzyme digestion and double enzyme digestion identification on the plasmid vector extracted from the bacterial liquid which is identified as positive by PCR, wherein restriction enzyme sites Xba I and Sma I introduced in the PCR process are selected as the restriction enzymes, and the enzyme digestion system is as follows: 2. mu.l of 10-fold Tango buffer, 10. mu.l of plasmid, 1. mu.l of Xba I, 1. mu.l of Sma I, 6. mu.l of enzyme-free water.

The preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold comprises the steps of A3, adopting Xba I and Sma I to carry out double enzyme digestion on pMD18-T-ech42 and pHT43 expression vectors respectively, and then connecting the expression vectors with enzyme digestion glue recovery products of chitinase gene ech42, wherein the connecting systems are pHT435 mul, ech 4212.8 mul, 10 XT 4DNA ligase buffer solution 2 mul and T4DNA ligase 0.2 mul.

The invention relates to a preparation method of genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, which comprises the following steps of:

b1, preparing a wild bacillus amyloliquefaciens BA electric transformation competent cell;

step B2, electric shock transformation, introducing the recombinant expression vector pHT43/ech42 into a bacillus amyloliquefaciens BA competent cell;

and step B3, screening the positive recombinant bacillus amyloliquefaciens, and extracting plasmids in the bacillus amyloliquefaciens.

The preparation method of the genetic engineering bacillus amyloliquefaciens for effectively preventing and treating the gray mold, disclosed by the invention, comprises the following steps of B1:

b1-1, taking the bacillus amyloliquefaciens BA strain stored in a laboratory out of a refrigerator at the temperature of-70 ℃, picking the bacterial liquid after melting, carrying out three-section marking on an LB solid culture medium, and carrying out inversion overnight culture in a constant-temperature incubator at the temperature of 37 ℃;

b1-2, picking a BA single colony growing after the overnight culture by using a yellow gun head, and culturing the single colony in 5ml of LB liquid culture medium at 150rpm and 37 ℃ in a shaking incubator overnight;

step B1-3, taking out 2.5ml of overnight culture by using a sterile blue gun head in an ultra-clean workbench, adding the overnight culture into 40ml of fresh Bacillus amyloliquefaciens growth culture medium, carrying out shaking culture at 37 ℃ and 200rpm until OD600 is 0.85-0.95, wherein the formula of the Bacillus amyloliquefaciens growth culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl and 91g/L of sorbitol;

step B1-4, immediately carrying out ice water bath on the bacterial liquid for 10min after the OD600 value is reached, subpackaging the bacterial liquid in 1.5ml centrifuge tubes, then centrifuging the bacterial liquid in a refrigerated centrifuge at 5000g and 4 ℃ for 5min, centrifuging each centrifuge tube twice, and collecting the bacillus amyloliquefaciens thalli;

step B1-5, mixing and re-suspending the enriched thalli by using an electric transfer culture medium without ions, sucking 1ml of the enriched thalli into a centrifugal tube each time, centrifuging the enriched thalli for 10min at 8500rpm at 4 ℃ through a low-temperature refrigerated centrifuge to enable the thalli to be gathered at the bottom of the tube, and rinsing the enriched thalli for 4 times by adopting the step, wherein the formula of the electric transfer culture medium without ions is 90g/L of sorbitol, 92.5g/L of mannitol and 100ml/L of glycerol;

and step B1-6, after washing, adding 60 mu L of electrotransformation culture medium into each centrifuge tube by using a sterile yellow spearhead to obtain the wild type bacillus amyloliquefaciens BA electrotransformation competent cells for later use, wherein the electrotransformation culture medium comprises 90g/L of sorbitol, 92.5g/L of mannitol and 100ml/L of glycerol.

The preparation method of the genetic engineering bacillus amyloliquefaciens for effectively preventing and treating the gray mold, disclosed by the invention, comprises the following steps of B2:

step B2-1, electrically transforming competent cells of the freshly prepared wild type bacillus amyloliquefaciens BA or the competence preserved in the refrigerator is taken out of the refrigerator, after the liquid of the competent cells is melted, the liquid of the competent cells is put on ice and mixed with 10 mul of recombinant plasmid pHT43/ech42 evenly, and then the mixture is subjected to ice water bath for 2 min;

step B2-2, transferring the ice-cooled mixture of step B2-1 to a 1mm electric shock cup, and placing the electric shock cup in a BIO-RAD GenePulser Xcell, and setting five gradients of electric transformation parameters: are respectively 12.5kv/cm, 15kv/cm, 17.5kv/cm, 20kv/cm, 21 kv/cm. After the parameters are set, the electric shock cup is immediately shocked, the electric shock resistance value is 200 omega, and the time constant of the electric shock is 4.5-5.0 ms;

b2-3, immediately sucking 1ml of resuscitation medium incubated at 37 ℃ by using a blue gun head after electric shock in the step B2-2, adding the resuscitation medium into an electric shock cup, uniformly mixing, transferring the mixture into a marked sterilized centrifuge tube, carrying out shaking culture on the centrifuge tube by using a culture box at 37 ℃ and 200rpm for 3-4h, wherein the prescription of the resuscitation medium is 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 90g/L of sorbitol and 70g/L of mannitol;

and B2-4 and B2-3, after shaking culture, centrifuging the centrifuge tube for 1min at 8000g, sucking 900 microliter of supernatant by using a gun head and discarding, re-suspending the thallus by using the residual liquid, coating the thallus into LB solid culture medium containing chloramphenicol, and culturing in a constant temperature incubator at 37 ℃ overnight.

The invention relates to application of genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, in particular to application of the genetically engineered bacillus amyloliquefaciens for preventing gray mold.

The genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating gray mold of the invention amplifies the chitinase gene ech42 of the gliocladium roseum by means of genetic engineering and is connected with an expression vector pHT43 to successfully construct a recombinant expression vector pHT43/ech 42. The wild strain and the genetically engineered bacillus amyloliquefaciens which are induced by IPTG for 8 hours and effectively prevent and treat gray mold are dissolved on cell walls by lysozyme, protein liquid is extracted, SDS-PAGE electrophoresis is carried out, and the genetically engineered bacillus amyloliquefaciens which contain pHT43/ech42 and effectively prevent and treat gray mold have target bands at 42 KD.

According to the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, a measured N-acetylglucosamine standard curve adopts a DNS reducing sugar method, and light absorption values of all tubes are respectively measured by N-acetylglucosamine standard solutions with different final concentrations under OD540, so that the standard curve shown in the following figure 3 is drawn. It can be seen that the N-acetylglucosamine content is in a linear function relationship with the absorbance, the obtained standard curve is y which is 0.5154x +0.0036, and the correlation coefficient of each point and the curve is R2 which is 0.9783, which shows that the correlation is very high.

The genetically engineered bacillus amyloliquefaciens for effectively preventing and treating the gray mold is compared with wild bacteria chitinase activity detection, the genetically engineered bacillus amyloliquefaciens pHT43-ech42/BA for effectively preventing and treating the gray mold is found to be respectively sampled after being induced by IPTG for 2h, 4h, 8h, 12h and 24h, meanwhile, the wild bacillus amyloliquefaciens BA is not induced by IPTG and is cultured for corresponding time, after the chitinase is measured by a DNS reducing sugar method, the enzyme activities of the genetically engineered bacillus amyloliquefaciens and the wild bacteria for effectively preventing and treating the gray mold are not invariable, the enzyme activity of the pHT43-ech42/BA strain is highest at the 8h of the chitinase, and the enzyme activity of the BA strain is highest at the 12h of the chitinase. The enzyme activity of the pHT43-ech42/BA strain in the 8 th hour is improved by about 54.3 percent compared with the enzyme activity of the BA strain in a wild strain, the genetically engineered Bacillus amyloliquefaciens pHT43/ech42 for effectively preventing and treating the gray mold is obviously improved compared with the wild strain in the aspect of preventing the gray mold, the disease prevention effect is still about 28.9 percent after 20 days of fungus spraying, and the biological control method has a lasting biological control effect.

Drawings

FIG. 1 is the construction of genetically engineered Bacillus amyloliquefaciens recombinant vector for effective prevention and treatment of gray mold;

FIG. 2 is a photograph showing the electrophoresis test of the target protein of genetically engineered Bacillus amyloliquefaciens for effective prevention and treatment of gray mold;

FIG. 3 is a standard curve of N-acetylglucosamine measured by the present invention;

FIG. 4 is the contrast curve of the detection of chitinase activity of genetically engineered Bacillus amyloliquefaciens and wild bacteria for effectively preventing and treating gray mold;

FIG. 5 is a photograph showing the comparison of the disease conditions of gray mold prevention by spraying genetically engineered Bacillus amyloliquefaciens, wild bacteria and water to tomatoes, wherein BA, ech42 and ck represent the spraying of wild bacteria and genetically engineered Bacillus amyloliquefaciens and water for effectively preventing gray mold, respectively;

FIG. 6 is a histogram of disease index of tomatoes sprayed with genetically engineered Bacillus amyloliquefaciens and treated with wild bacteria to prevent gray mold according to the present invention;

FIG. 7 is a histogram comparing the control effect of the genetically engineered Bacillus amyloliquefaciens sprayed to tomatoes treated with wild bacteria to prevent gray mold.

Detailed Description

The first embodiment is as follows:

The genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating gray mold is constructed as shown in the attached drawing 1, and a chitinase gene ech42 of Gliocladium roseum is amplified by means of genetic engineering and is connected with an expression vector pHT43 to successfully construct a recombinant expression vector pHT43/ech 42.

The gene engineering bacillus amyloliquefaciens for effectively preventing and treating gray mold has target protein electrophoresis inspection pictures as shown in the attached figure 2: wherein the M-protein molecular mass standard; 1-IPTG induction of Bacillus amyloliquefaciens containing pHT43/ech 42; 2-does not induce bacillus amyloliquefaciens containing pHT43/ech42 with IPTG; 3-IPTG induced Bacillus amyloliquefaciens; 4-Bacillus amyloliquefaciens not induced with IPTG. From the figure, it can be obtained that wild bacteria after 8h of IPTG induction and genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating gray mold dissolve cell walls by lysozyme, extract protein liquid, and perform SDS-PAGE electrophoresis, the electrophoresis result is shown in figure 2, and the genetically engineered Bacillus amyloliquefaciens containing pHT43/ech42 for effectively preventing and treating gray mold has target bands at 42 KD.

The standard curve of the measured N-acetylglucosamine of the genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating gray mold is shown in the attached figure 3: the absorbance of each tube was measured by the DNS reducing sugar method using N-acetylglucosamine standard solutions of different final concentrations at OD540, and the standard curve shown in FIG. 3 was plotted. It can be seen that the N-acetylglucosamine content is in a linear function relationship with the absorbance, the obtained standard curve is y which is 0.5154x +0.0036, and the correlation coefficient of each point and the curve is R2 which is 0.9783, which shows that the correlation is very high.

The detection contrast curve of the activity of the genetically engineered bacillus amyloliquefaciens and the chitinase of the wild bacteria for effectively preventing and treating the gray mold is shown in the attached drawing 4: the genetically engineered Bacillus amyloliquefaciens pHT43-ech42/BA for effectively preventing and treating gray mold is respectively sampled after 2h, 4h, 8h, 12h and 24h of IPTG induction, meanwhile, wild Bacillus amyloliquefaciens BA is not induced by IPTG, cultured for corresponding time, and tested for chitinase by a DNS reducing sugar method, and the result is shown in figure 4. It can be seen that the enzyme activities of genetically engineered Bacillus amyloliquefaciens and wild bacteria for effectively preventing and treating gray mold are not invariable, the enzyme activity of chitinase of pHT43-ech42/BA strain is highest in the 8 th hour, and the enzyme activity of chitinase of BA strain is highest in the 12 th hour. The enzyme activity of pHT43-ech42/BA strain at the 8 th hour is improved by about 54.3 percent compared with the enzyme activity of BA of a wild strain.

The genetic engineering bacillus amyloliquefaciens spray application, the wild bacteria spray application and the water spray application on the tomato for effectively preventing the gray mold disease potting experiment comprise the following steps:

(1) placing tomato seedlings growing to 5 leaves and 1 heart in a gardening station into a self-made plastic shed, selecting the tomato seedlings with consistent sizes, randomly dividing the tomato seedlings into four groups, wherein 31 seedlings in each group are placed in the shed and then are required to be slow-released for about 5 days, so that the seedlings adapt to a new environment;

(2) the original strain cultured in a constant temperature shaking incubator at 37 ℃ and the genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating gray mold are respectively diluted by 10 times and are used for spraying seedlings. Adding 100ml of tap water into the tomato full of botrytis cinerea, and fully shaking up to prepare a bacterial suspension of the tomato botrytis cinerea;

(3) the disease prevention effect is divided into 3 groups: spraying original strain BA, the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold and CK (distilled water);

(4) after the biocontrol strain is sprayed (step three), the bacterial suspension of the botrytis cinerea is sprayed again after 24 hours;

(5) disease indices were determined on

days

15 and 20 after spraying the biocontrol strain.

A comparative photograph of the disease condition of preventing gray mold by spraying genetically engineered bacillus amyloliquefaciens for effectively preventing gray mold, spraying wild bacteria and water on tomatoes is shown in the attached drawing 5, wherein BA, pHT43/ech42 and ck respectively represent spraying wild bacteria, genetically engineered bacillus amyloliquefaciens for effectively preventing gray mold and water, and pot experiments respectively determine corresponding disease condition indexes and prevention effects on

days

15 and 20. In the aspect of preventing gray mold, the genetically engineered bacillus amyloliquefaciens pHT43/ech42 for effectively preventing gray mold is obviously improved compared with wild bacteria, the disease prevention effect is still about 28.9 percent after 20 days of fungus spraying, and the disease index is shown in table 1:

TABLE 1 Pot culture experiment illness state comparison table

The disease index histogram of tomatoes which are sprayed with genetically engineered bacillus amyloliquefaciens and treated by wild bacteria and are used for effectively preventing and treating gray mold in the embodiment is shown in the attached drawing 6, the attached drawing 7 is a comparison histogram of the disease control effects of tomatoes which are sprayed with genetically engineered bacillus amyloliquefaciens and treated by wild bacteria and are used for preventing and treating gray mold in the embodiment, the biocontrol effect in the embodiment can be obviously better seen from the attached drawings 6 and 7, and the disease control effects of the tomatoes which are treated by different methods are shown in the table 2:

TABLE 2 comparison table of the disease prevention effect of the potted plant experiment

The second embodiment is as follows:

according to a specific embodiment, the preparation method of the genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating the gray mold comprises the following steps of:

step A, amplifying chitinase gene ech42 of Gliocladium roseum through genetic engineering, connecting with Bacillus amyloliquefaciens expression vector pHT43, constructing recombinant expression vector pHT43/ech42,

and step B, introducing the recombinant expression vector pHT43/ech42 in the step 1 into Bacillus amyloliquefaciens by setting electric shock transformation, wherein the electric shock voltage is 12.5kv/cm or 15kv/cm or 17.5 kv/cm.

The third concrete implementation mode:

according to the first or second embodiment, the preparation method of the genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating the gray mold comprises the following steps:

step a1, obtaining c.rosea chitinase gene ech42 by PCR cloning;

In the preparation method of genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, a gene sequence of c.rosea chitinase ech42 gene (DQ523687) is found through GenBank in step a1, a signal peptide sequence is detected by using signal peptide detection software, a cloned gene does not contain a restriction enzyme site introduced into a primer, and the primer is designed by using primmer5 software:

In the preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, a PCR amplification product in the step A2 is recovered and purified by a reference Kit StarPrep Gel Extraction Kit; connecting the recovered target gene ech42 with a T vector, wherein the reaction system is 1 mul of pMD18-T vector, 1 mul of target gene ech424 mul and 5 mul of solution I; and (3) carrying out single enzyme digestion and double enzyme digestion identification on the plasmid vector extracted from the bacterial liquid which is identified as positive by PCR, wherein restriction enzyme sites Xba I and Sma I introduced in the PCR process are selected as the restriction enzymes, and the enzyme digestion system is as follows: 2. mu.l of 10-fold Tango buffer, 10. mu.l of plasmid DNA, 1. mu.l of Xba I, 1. mu.l of Sma I, and 6. mu.l of water without enzyme.

In the method for preparing genetically engineered bacillus amyloliquefaciens capable of effectively preventing and treating gray mold according to the embodiment, a PCR product Gel recovery reference Kit (StarPrep Gel Extraction Kit) in the step A2 is carried out, before Gel recovery, a clean and sterile Gel cutting blade and an ultraviolet Gel analyzer are required to be prepared, 1% agarose Gel is required to be prepared, and a new electrophoresis solution (1 × TAE) is required to be replaced, and the method comprises the following steps:

(1) cutting the glue: preparing a gel plate, carrying out spotting electrophoresis for about 20min, taking out the gel plate, rapidly cutting a target gene fragment under an ultraviolet lamp, removing the part without DNA at the edge of the gel block as much as possible, and rapidly operating under the ultraviolet lamp to reduce the damage of ultraviolet and the like to DNA in order to facilitate the gel block to be cut as much as possible;

(2) weighing the mass of the rubber block: weighing 1.5ml of centrifuge tube in advance, peeling, repeatedly cutting the cut DNA fragments to make the DNA fragments cut into the tube, wherein the higher the DNA content of the gel block, the better the DNA content is, and the DNA content is not too heavy;

(3) sol: according to the following steps: 1 proportion, adding 1 mul of sol solution (MB) into 1mg of the gel, incubating in a water bath at 55 ℃, and slightly inverting the centrifuge tube every 1-2 minutes to ensure that the gel block is fully contacted with the sol solution, wherein the gel block is gently inverted to prevent the DNA fragments from being broken;

(4) DNA binding: cooling the liquid after sol to room temperature, transferring the liquid to a centrifugal adsorption column with a collecting pipe, standing for 1-2min at room temperature, balancing, placing the liquid in a centrifugal machine, centrifuging at 12000rpm for 1min, carefully sucking the liquid in the collecting pipe into the adsorption column again after centrifuging to ensure the highest recovery rate, placing the liquid in the collecting pipe into the collecting pipe, centrifuging at 12000rpm for 1min again, and discarding the waste liquid in the collecting pipe after centrifuging;

(5) and (3) column cleaning: adopting cleaning solution (MW) added with absolute ethyl alcohol, measuring 600 mul by a pipette, adding the pipette on an adsorption column containing a DNA sample, centrifuging for 1min at 10000rpm of a high-speed centrifuge, pouring off waste liquid at the bottom of the tube, inserting the column into the tube again, and waiting for next centrifugation;

(6) the column was washed again: measuring 600 mu l (MW) by using a pipette, adding the MW into a column, centrifuging at the same rotating speed as the steps, pouring the waste liquid at the bottom of the column after centrifuging, inserting the column into a return pipe, carrying out air separation for 4min, throwing away a collecting pipe after the air separation is finished, airing the column in a dry place, and volatilizing absolute ethyl alcohol to prevent influence on subsequent enzyme digestion and enzyme linked operation;

(7) and (3) elution: taking a sterilized 1.5ml centrifuge tube, marking, placing the adsorption column in it, adding 55 μ l deionized and sterilized water with temperature of 65 deg.C into the adsorption film at the center of the centrifugal adsorption column, standing at room temperature for 1min, and centrifuging at 12000rpm for 1 min. After centrifugation, carefully sucking the liquid in the centrifugal tube into the adsorption column and centrifuging again through the column so as to improve the recovery rate;

(8) and (3) storage: the adsorption column was discarded and the gel recovered fragments were stored in a-20 ℃ freezer for future use.

The preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating the gray mold comprises the following steps of preparing the competence of escherichia coli E.coli DH5 alpha in the step A2:

(1) an original E.coli DH5 alpha strain stored in a refrigerator at the temperature of-70 ℃ is dipped by a strain inoculating ring, and streaking is carried out on an LB solid plate by adopting a three-section streaking mode. Placing the scribed flat plate into a constant-temperature incubator to be cultured at the temperature of 37 ℃;

(2) observing a single colony of E.coli DH5 alpha growing on the flat plate after overnight, adopting a mode of inoculating a bacterium liquid culture, picking the single colony on the flat plate by using a sterile yellow gun head, amplifying the single colony in 5ml of LB liquid culture medium, and oscillating the single colony in a shaking table at 37 ℃ overnight, wherein the E.coli DH5 alpha grows to the late logarithmic growth stage;

(3) sucking 1ml of shake-cultured bacterial liquid, transferring the liquid to 50ml of fresh LB culture medium, shake-culturing the liquid for 2 hours at a constant temperature of 180rpm and 37 ℃, measuring the OD600 value of the bacterial liquid every half an hour until the OD600 is about 0.5, and stopping shaking the bacterial liquid;

(4) subpackaging the cultured bacteria liquid into 1.5ml centrifuge tubes, adding 1250 mul of bacteria liquid into each tube, placing the subpackaged bacteria liquid on ice for ice bath for 10min, placing the centrifuge tubes containing the bacteria liquid on a refrigerated centrifuge, centrifuging at 3000g at 4 ℃ for 10min, and enriching E.coli DH5 alpha;

(5) after centrifugation, the supernatant was decanted off, and 500. mu.l of 0.1mol/L CaCl2 solution after ice-bath was aspirated by a blue-gun head to resuspend the cells. After the resuspended thalli is subjected to ice bath for 20min, centrifuging for 10min at the temperature of 4 ℃ and the rotating speed of 3000g again;

(6) after centrifuging the centrifuge tube, the Escherichia coli can be obviously observed to be enriched at the bottom of the tube, the supernatant is discarded, if the Escherichia coli is not discarded completely, the Escherichia coli can be sucked out by a yellow or white gun head, then 200 mu L of 0.05mol/L CaCl2 solution containing 15% of glycerol is added, the step needs to be carried out on ice to prevent the influence on the competent transformation effect, and finally the Escherichia coli is ice-bathed for 2min and placed into a refrigerator at the temperature of-70 ℃ to be taken at any time during transformation.

The preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating the gray mold, which is provided by the embodiment, comprises the following steps of transforming a connecting product into a competence of escherichia coli E.coli DH5 alpha in the step A2:

(1) taking out the separately packaged and stored competent cells in a refrigerator with a glove at the temperature of-70 ℃, unfreezing the competent cells at room temperature, and immediately placing the competent cells on ice;

(2) completely transferring products subjected to T-vector connection in the steps to escherichia coli competent cells by adopting a white gun head with a total measuring range of 10 mu l, uniformly mixing the mixed solution by using a gun in a proper condition after the products are injected, and carrying out ice-water bath for 30 min;

(3) immediately putting the bacterium solution subjected to ice bath into a metal bath at 42 ℃ for heat shock for 90s, and quickly cooling on ice for 3min after the heat shock is finished;

(4) adding 1ml of fresh LB liquid medium (without antibiotics) into the heat shocked centrifuge tube in a superclean workbench, shaking and culturing for 1 hour at 37 ℃ in a shaking table (the shaking time can be properly prolonged), so that escherichia coli can normally grow, and an antibiotic resistance gene stored in a plasmid is expressed;

(5) balancing a centrifugal tube, performing high-speed centrifugation on the centrifugal tube to deposit thalli, discarding 1ml of supernatant in a super clean bench, gently suspending and uniformly mixing the residual 200 mu l of supernatant and thalli by using a blue gun head, coating the residual bacterial suspension on an LB solid culture medium containing Amp by using a coating rod sterilized on an alcohol lamp, and performing inverted culture in a 37 ℃ constant-temperature incubator for overnight after bacterial liquid is dried;

(6) a control is needed to be made during the transformation, the same volume of sterile double distilled water is used for replacing the connection product, other operations are the same, and the control is respectively coated on an LB solid culture medium containing Amp and an LB solid culture medium without Amp and used as a control to determine the activity and the competence activity of the Amp antibiotic.

In the preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, the identification steps of the recombinants in the step A2 are as follows, and the table 1 is a bacterial liquid PCR reaction system table:

(1) counting and marking single colonies growing in the plate simply, then selecting 4 single colonies, inoculating the single colonies into a liquid culture medium for shake culture overnight, and adding Amp antibiotics into the liquid culture medium to enhance the screening effect on recombinants;

(2) after overnight culture, 1. mu.l of the bacterial solution was subjected to PCR in a 20. mu.l system, as shown in tables 2 to 5, under the same amplification conditions as above;

(3) after the program of the PCR instrument is finished, 5 mul of bacteria liquid PCR product is extracted and mixed with 1 mul of loading buffer, the mixture is spotted in prepared 1% agarose gel, electrophoresis is carried out under the condition of 180v voltage, whether fragments with the size consistent with that of target gene ech42 are contained in the corresponding bands of a Marker or not is observed, and if positive, the bacteria liquid can be subjected to the next plasmid extraction work.

TABLE 1 bacterial liquid PCR reaction system

In the method for preparing genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold according to the embodiment, the method for extracting escherichia coli plasmids in the step A2 is carried out by referring to an escherichia coli Plasmid extraction reference Kit (StarPrep Plasmid Miniprep Kit), and the steps are as follows:

(1) preparing: adding absolute ethyl alcohol and RNase A into respective mother liquor according to the relevant instructions of the kit, uniformly mixing and marking for later use;

(2) and (3) collecting thalli: respectively taking 4 clean sterilized centrifuge tubes, marking, respectively adding 1.5ml of overnight cultured bacterial liquid, centrifuging at 12000rpm for 1min, centrifuging, removing the supernatant, if the enriched bacterial liquid at the tube bottom is less, adding 1.5ml of bacterial liquid again, centrifuging again, and removing the supernatant (the centrifugation times depend on the concentration of the bacterial liquid);

(3) suspension of the bacteria: adding 250 mu l of cell suspension (S1) into the centrifuge tube after bacteria collection, and repeatedly blowing and beating the centrifuge tube by using a blue gun head to ensure that the enriched bacteria are fully suspended in the S1 solution;

(4) cracking and neutralizing: adding 250 ul (S2) into the centrifuge tube after the above steps, immediately covering the tube cover, slightly reversing for several times to mix the materials evenly, placing the mixture for standing until the bacteria liquid is clarified, adding 350 ul (S3) and slightly mixing the mixture evenly, placing the mixture in a centrifuge for high-speed centrifugation for 15min, and observing white solid matters at the bottom of the tube after centrifugation;

(5) DNA membrane binding: and taking out the centrifuged centrifuge tube, carefully sucking out the supernatant by using a blue gun head, transferring the supernatant into a marked centrifugal adsorption column inserted into the collection tube, centrifuging at 12000rpm at room temperature for 1min, and sucking the liquid in the collection tube into the adsorption column again and inserting the liquid into the collection tube to ensure the maximum recovery efficiency. Centrifuging at 12000rpm for 1min at room temperature again, and discarding the waste liquid in the collecting pipe;

(6) cleaning an adsorption column: adding 500 μ l (WB) into the adsorption column, covering with tube cover, centrifuging at high speed in a centrifuge, and discarding the waste liquid in the tube. Adding 500 mul (WB) into the adsorption column again, centrifuging again at the same rotating speed, opening the adsorption column cover without adding any liquid after centrifuging, placing the adsorption column into a centrifuge for no-load centrifugation for 2min, and drying in a drying place for about 15min after centrifuging, so as to carry out the next operation;

(7) and (3) eluting the plasmid: drying the column, placing into a clean centrifuge tube, adding 55 μ l sterilized deionized water, and standing in 60 deg.C water bath at room temperature for 2min to increase recovery rate. The plasmid DNA was collected by centrifugation at 12000rpm for 1min at room temperature. In order to ensure the recovery efficiency, the centrifuged liquid containing the plasmid DNA is sucked out again and put on a silica gel adsorption membrane for secondary centrifugation;

(8) and (3) plasmid preservation: the centrifugal adsorption column was discarded, and the plasmid solution was stored in a-20 ℃ freezer and taken at any time.

In the preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating the gray mold, the substep of cutting and detecting positive clones by the plasmid in the step A2 is as follows:

(1) and (3) carrying out single enzyme digestion and double enzyme digestion identification on the plasmid vector extracted from the bacterial liquid which is identified as positive by PCR, wherein the restriction enzyme is restriction enzyme sites (Xba I and Sma I) introduced in the PCR process. The following 20. mu.l system was selected (the same applies to the single cleavage system) as in Table 2 below, with reference to the Thermo restriction enzyme insert specifications:

TABLE 2 enzyme digestion System

To ensure the efficiency of the digestion, the centrifuge tubes were incubated overnight at 37 ℃ in a metal bath.

(2) The following day, the directly extracted plasmid, the single digested plasmid and the double digested plasmid were mixed with loading buffer in sequence, and 5. mu.l was spotted and subjected to electrophoresis in 1% agarose gel at 180v for 15 min. And observing under an ultraviolet lamp after electrophoresis, wherein under the condition of three lanes, the first lane with the plasmid without enzyme digestion is lower than the second lane with the plasmid with single enzyme digestion, the lane with the plasmid with double enzyme digestion shows two cleaned bands, the last lane is lower, and if the size of the lower small DNA fragment is matched with that of the target gene, enzyme digestion is identified as positive.

(3) Sequencing of the recombinant T vector: and (3) sending the escherichia coli liquid with the correct T carrier in the PCR and single-double enzyme digestion detection connection of the liquid to a sequencing company for sequence determination, splicing after a sequencing result is obtained, and comparing the splicing result through comparison service on NCBI.

In the preparation method of genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, plasmid extraction in the step A3 is implemented by respectively inoculating sequencing-completed escherichia coli containing DH5 alpha of pMD18-T-ech42 and escherichia coli containing pHT43 expression vectors into LB culture medium containing Amp antibiotics, carrying out shake culture, and respectively carrying out plasmid extraction operation on bacterial liquid by referring to the escherichia coli plasmid extraction method after shake culture.

The preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold and the double enzyme digestion method of the plasmid vector adopt Xba I and Sma I to respectively carry out double enzyme digestion on pMD18-T-ech42 and pHT43 expression vectors, the temperature of the expression vectors is raised overnight at 34 ℃ in a metal bath, the enzyme digestion system is 50 mu l, and the system is as follows in the following table 3:

TABLE 350. mu.l of digestion System

In the preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, after enzyme digestion of the gel recovery target gene and the expression vector enzyme digestion segment in the step A3 is finished, the enzyme digestion product is spotted in 1% agarose gel, gel recovery is carried out by referring to a method for recovering and purifying a PCR amplification product, a target gene at the lower end is recovered by a lane gel containing a segment (ech42) of the target gene, and a vector segment at the upper end is recovered by a lane gel containing an expression vector pHT 43.

In the preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold, Xba I and Sma I are adopted to carry out double enzyme digestion on pMD18-T-ech42 and pHT43 expression vectors respectively in the step A3 in the step A3, then the expression vectors and enzyme digestion glue recovery products of chitinase gene ech42 are connected, and the connector systems are pHT435 mul, ech 4212.8 mul, 10 XT 4DNA Ligase Buffer 2 mul and T4DNA Ligase 0.2 mul.

The fourth concrete implementation mode:

according to the first or second embodiment, the preparation method of the genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating the gray mold comprises the following steps of:

In the preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating the gray mold, the step B1 comprises the following steps:

In the preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating the gray mold, the step B2 comprises the following steps:

In the preparation method of the genetically engineered bacillus amyloliquefaciens for effectively preventing and treating the gray mold, the step B3 of screening and identifying the positive recombinants comprises the following steps:

(1) picking up a single colony after overnight culture and adding the single colony into LB culture medium with chloramphenicol, and carrying out shaking culture at 37 ℃ and 180rpm for overnight;

(2) bacterial liquid PCR: according to the method for detecting the positive recombinant bacillus amyloliquefaciens by the people such as the wild goose positive soldier and the like, 10mg/ml lysozyme solution is prepared and added into 1ml of bacterial liquid cultured with the bacillus amyloliquefaciens till the final concentration is 0.4mg/ml, the lysozyme is fully exerted by oscillating for 30min at 37 ℃ of a shaking table, the cell wall of the bacillus amyloliquefaciens is dissolved, the bacterial liquid is boiled in boiling water, then the bacterial liquid is immediately put into a refrigerator at 20 ℃ below zero for freezing for 5min to fully break the cell wall, plasmids are dissolved out, then 1 mu l of lysate is taken for carrying out bacterial liquid PCR detection, and the PCR system is 20 mu l. Carrying out PCR verification on the bacterial liquid to obtain positive bacterial liquid, and carrying out subsequent extraction on plasmids;

(3) and (3) extracting plasmids and carrying out enzyme digestion detection: plasmid extraction in Bacillus amyloliquefaciens refers to the method for extracting Escherichia coli plasmid, and is characterized in that lysozyme is added into a cell suspension (S1) to a final concentration of 0.4mg/ml, and the lysozyme is fully exerted by shaking for 30min at 37 ℃ of a shaking table. The subsequent steps are the same as the previous plasmid extraction. After extracting the plasmid, carrying out single enzyme digestion and double enzyme digestion identification on the plasmid, wherein the enzyme digestion system is 20 mu l, then spotting in 1% agarose gel, and observing the result in an ultraviolet gel imaging system after electrophoresis.

The fifth concrete implementation mode:

according to the preparation method of the genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating the gray mold, the induced expression of the chitinase and the SDS-PAGE electrophoresis thereof comprise the following steps:

(1) preparation of reagents

TABLE 4 formulation of concentrated and fractionated gums

(2) Preparation of the protein of interest

Inoculating bacillus amyloliquefaciens BA transferred into a recombinant plasmid pHT43/ech42 into a2 bottle 50ml LB culture medium containing chloramphenicol by using a yellow gun head, carrying out constant temperature shaking culture in a 37 ℃ constant temperature shaking culture box at 180rpm, detecting the OD value every half hour, suspending shaking culture until the OD600 is 0.6-0.8, adding IPTG with the final concentration of 1mmol/L into the bacillus amyloliquefaciens BA with the pH T43/ech42, inducing at 37 ℃ and 180rpm for 8 hours, adding lysozyme with the final concentration of 0.4mg/ml into a bacterial liquid, and cracking the bacillus amyloliquefaciens. And after the bacterial liquid is clarified, respectively filling the bacterial liquid into 1.5ml centrifuge tubes for later use. Before sample application, 16 mul of bacterial liquid is taken out, 4 mul of reduced 5 xSDS sample loading buffer solution is added, after uniform mixing, boiling is carried out for 10min, and protein sample is finished and can be directly added into polyacrylamide gel.

(3) Preparing gel, comprising the following steps:

(3-1) cleaning the glass plate: and cleaning the four glass plates with glue by using tap water, washing the four glass plates by using distilled water once, and airing the four glass plates in a dry place.

And (3-2) clamping the glass plate and the glue maker, putting the glass plate on a rack, filling distilled water, and detecting whether liquid leaks or not.

(3-3) after no liquid leakage is observed, 10% separation gel is prepared and is filled, two plates are filled with gel, distilled water is slightly added through a gun head after the gel is filled, and whether the liquid level of the distilled water is reduced or not can be observed to prevent the liquid leakage.

(3-4) after about 30min, the separation gel is solidified, at the moment, the upper layer of distilled water is poured out, 5% of concentrated gel is prepared, and the gel is filled and inserted into a comb, so that bubbles are not generated in the process of inserting the comb as much as possible. After about 30min the concentrated gel solidifies and the spots can be applied to the gel wells.

(4) Spotting is carried out

The prepared rubber plate is taken off the base, placed in an electrophoresis tank, and added with 1 XTirs-glycine electrophoresis buffer solution, the added solution is soft to prevent bubbles from generating at the bottom of the rubber plate and affecting the electrophoresis effect, and the liquid level is added to the rubber-impregnated hole. After the electrophoresis solution was added, a protein Marker (5. mu.l) and a boiled protein sample (20. mu.l) were sequentially added.

(5) Electrophoresis

When bromophenol blue is displayed in the upper concentrated gel, electrophoresis is carried out by adopting a voltage of 80 v; when the protein runs to the interface of the separation gel and the concentration gel, the protein is compressed into a line, and at this time, 120v voltage is selected for electrophoresis.

(6) Dyeing process

Bromophenol blue as an indicator can represent a small protein band in the glue running process, when the blue band runs to the position about 1cm away from the bottom of a glass plate, an electrophoresis apparatus can be closed to stop electrophoresis, after an upper layer of concentrated glue is cut off by a clean blade, a lower layer of separated glue is soaked into prepared Coomassie brilliant blue staining solution for staining. And taking out the separation gel after 1h, boiling and decoloring by using weak decoloring liquid until a clear strip is displayed on the gel plate, putting the gel plate into a gel imaging system for imaging and storing, and finally putting the gel plate into a preserving liquid for preserving.

The sixth specific implementation mode:

according to the first or second embodiment, the method for preparing genetically engineered Bacillus amyloliquefaciens for effectively preventing and treating gray mold comprises the following steps of measuring the activity of chitinase by a DNS reducing sugar method, wherein the DNS reducing sugar method is a dinitrosalicylic acid method, the method has the principle that dinitrosalicylic acid (DNS) can perform oxidation-reduction reaction with reducing sugar, and can display different shades of color according to the content of the reducing sugar after boiling, the chitinase enzymatic hydrolysis product is reacted with the dinitrosalicylic acid by using the principle, and the color is compared in an enzyme labeling instrument after boiling to obtain the activity of the chitinase, and the method comprises the following steps of:

(1) preparation of Standard Curve

Adding corresponding reagents according to the reagent contents in the table 5, boiling for 10min after fully and uniformly mixing, then immediately cooling by using flowing cold water, fixing the volume of each tube to 10ml, using distilled water as a blank, measuring the light absorption value of each tube at the wavelength of 540nm by using an enzyme-labeling instrument, recording, repeating the steps for three times for each tube, and calculating the average value of the three times. And drawing an N-acetylglucosamine standard curve by using the N-acetylglucosamine content as an abscissa and the light absorption value as an ordinate.

TABLE 5 preparation of acetylglucosamine Standard Curve

(2) Inducible expression of recombinant chitinase

Inoculating bacillus amyloliquefaciens BA transferred into a recombinant plasmid pHT43/ech42 into a2 bottle 100ml LB liquid culture medium containing chloramphenicol by using a yellow gun head, simultaneously inoculating original bacillus amyloliquefaciens strain (BA) without exogenous plasmid into a 100ml LB liquid culture medium, carrying out shaking culture at 180rpm in a 37 ℃ constant temperature shaking culture box, detecting an OD value every half hour until the OD600 is 0.6-0.8, suspending shaking culture, adding IPTG with the final concentration of 1mmol/L into the bacillus amyloliquefaciens containing the recombinant plasmid, respectively carrying out induction at 37 ℃ and 180rpm for 2h, 4h, 8h, 12h and 24h, and respectively carrying out sampling.

(3) Determination of recombinant chitinase Activity

Centrifuging the bacterial liquid at different periods after IPTG induction for 1min by using a centrifuge 8000rpm, respectively sucking 0.5mL of supernatant into a test tube (the control is the supernatant after boiling and inactivation at 100 ℃ and is taken as the control), adding 1.0mL of 1% (w/v) colloidal chitin, incubating for 1h at 37 ℃ in a water bath kettle, after the water bath is finished, recovering to room temperature, adding 1.5mL of DNS reagent, quickly boiling for 10min at 100 ℃, immediately cooling by using tap water after the water bath is finished, centrifuging for 5min by using the centrifuge at 8000rpm, fixing the volume of the reaction liquid to 10mL by using distilled water, taking the supernatant into an enzyme labeling instrument, measuring the absorbance at the wavelength of 540nm, and repeating the treatment for three times.

Sequence listing

<110> northeast university of agriculture

<120> genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold as well as preparation method and application thereof

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1218

<212> DNA

<213> Gliocladium roseum (Bionectria ochroleuca)

<400> 1

cgtgccactc ctcgtatgga ggacctggct tccactgacc tttctacgcg tgcaactgga 60

tccgtcaatg ctgtttactt cactaactgg ggcatttacg gacgcaactt ccagcctgcg 120

gatcttccag cttcaagcat ttcgcatgtc ctctattctt tcatgaacct ccgagcggat 180

ggcacggttt actcgggtga cacctacgcc gacttggaga agcattactc tgacgactca 240

tggaatgata tcggaacaaa cgcctatggt tgtgtcaagc agctctacaa gctcaagaag 300

gcaaaccgct cgctcaagat catgctgtcc attggtggct ggacttggtc gaccaacttt 360

cccgctgctg cctccaccga ggctacccgt gctacatttg ccaagaccgc tgttgaattc 420

atgaaggatt ggggttttga cggcattgac gtcgactggg agtaccccgc cagtgagaca 480

gaagccaaca acatggtcct ccttcttcag cgagttcgcc aggagctcga ctcgtactcc 540

gcaacatatg ctaatggcta tcacttccaa ctttccattg ccgctcccgc aggacctgac 600

cattacaagg ttcttaagct agcccagctc ggttccgtcc tcgacaacat caacctcatg 660

gcctacgact atgcaggttc ctgggacagt gtcagcggtc atcaagccaa cctgtatcct 720

agcacatcaa accccagctc aactccattc agcaccaagg ctgcggtcga cgcatacatc 780

gcagctggcg tccctgcaag caagatcatt ctaggtatgc ccatctacgg cagagctttt 840

gtgggaaccg acggaccagg caagccctac tccactatcg gcgaaggcag ctgggagagc 900

ggaatctggg actacaaagt ccttcccaag gccggcgcaa ccgtgattac cgactccgcg 960

gccggtgcta cctacagtta cgactccagc agcaggacca tgatctcata cgataccccc 1020

gatatggtcc gcacaaaggt ctcatatgct aagggtctcg gtctcggagg cagcatgttc 1080

tgggaggcat cagccgacaa gaccggctct gactcgctta tcggcactgc cctcagcagc 1140

atgggcagcc ttgacagcac ccagaactgc ctcagctacc ccaactccaa gttcgacaat 1200

atcaagaaca gcctctcc 1218

Claims

1. A genetically engineered bacillus amyloliquefaciens for effectively preventing and treating gray mold is characterized in that: the strain is classified as Bacillus amyloliquefaciens, chitinase genes of Gliocladium roseum are transferred into the strain, and the strain is preserved in China microorganism culture Collection center (CGMCC) in 2017, 6 and 29 days, and the preservation number is CGMCC No. 14366.

2. A preparation method of genetically engineered Bacillus amyloliquefaciens for effectively controlling gray mold as claimed in claim 1, which is characterized in that: the method comprises the following steps:

and step B, introducing the recombinant expression vector pHT43/ech42 into Bacillus amyloliquefaciens BA by setting electric shock transformation, wherein the electric shock voltage is 12.5kv/cm or 15kv/cm or 17.5 kv/cm.

3. The preparation method of genetically engineered bacillus amyloliquefaciens for effectively controlling gray mold as claimed in claim 2, characterized in that: the step A comprises the following steps:

step a1, obtaining c.rosea chitinase gene ech42 by PCR cloning;

step A3, connecting the chitinase gene ech42 obtained in step A2 with an expression vector pHT 43.

4. The preparation method of genetically engineered Bacillus amyloliquefaciens for effectively controlling gray mold as claimed in claim 3, wherein the bacillus amyloliquefaciens is prepared from the following raw materials in parts by weight: found by GenBank in step A1C.roseaChitinaseech42The gene sequence of gene DQ523687, signal peptide detection software is used to detect the signal peptide sequence, and the cloned gene does not contain the enzyme cutting site introduced on the primer, and the primer is designed by using the primmer5 software:

primer and method for producing the sameech42E：5'-TATCTAGACGTGCCACTCCTCGTATGGAGG-3', the restriction enzyme cutting site Xba I;

ech42b: 5'-CCCCCCGGGGGAGAGGCTGTTCTTGAT-3', the restriction enzyme cutting site Sma I;

the mixture of the PCR reactions was: template 1 mu l, 10 mu M upstream primerech42E1 ul, 10 uM downstream primerech42B1 mul, 5 mul of 10 multiplied EasyTaq buffer solution, 2.5mMdNTPs4 mul, 0.5 mul of EasyTaq DNA polymerase and 37.5 mul of ultrapure water;

5. The preparation method of genetically engineered Bacillus amyloliquefaciens for effectively controlling gray mold as claimed in claim 3, wherein the bacillus amyloliquefaciens is prepared from the following raw materials in parts by weight: recovering and purifying the PCR amplification product in the step A2 by using a reference kit StarPrepGelextraction kit; connecting the recovered target gene ech42 with a T vector, wherein the reaction system is 1 mul of pMD18-T vector, 1 mul of target gene ech424 mul and 5 mul of solution I; and (3) carrying out single enzyme digestion and double enzyme digestion identification on the plasmid vector extracted from the bacterial liquid which is identified as positive by PCR, wherein restriction enzyme sites Xba I and Sma I introduced in the PCR process are selected as the restriction enzymes, and the enzyme digestion system is as follows: 2 mul of 10 times of Tango buffer solution, pMD18-T/ech4210 mul, Xba I1 mul, Sma I1 mul and 6 mul of enzyme-free water.

6. The preparation method of genetically engineered Bacillus amyloliquefaciens for effectively controlling gray mold as claimed in claim 3, wherein the bacillus amyloliquefaciens is prepared from the following raw materials in parts by weight: in step A3 Xba I and Sma I were used to treat pMD18-T-ech42Carrying out double enzyme digestion on the expression vector pHT43, and then carrying out double enzyme digestion on the expression vector and the chitinase geneech42The enzyme digestion glue recovery products are connected, the connecting system is pHT435 mu l,ech4212.8 μ l, 2 μ l of 10 XT 4DNA ligase buffer, 0.2 μ l of T4DNA ligase.

7. The preparation method of genetically engineered bacillus amyloliquefaciens for effectively controlling gray mold as claimed in claim 2, characterized in that: the step B comprises the following steps:

8. The method for preparing genetically engineered bacillus amyloliquefaciens for effectively controlling gray mold as claimed in claim 7, wherein the method comprises the following steps: step B1 includes the following steps:

step B1-3, taking out 2.5ml of overnight culture by using a sterile blue gun head in an ultra-clean workbench, adding the overnight culture into 40ml of fresh bacillus amyloliquefaciens growth culture medium, carrying out shaking culture at 37 ℃ and 200rpm until OD600=0.85-0.95, wherein the formula of the bacillus amyloliquefaciens growth culture medium comprises 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl and 91g/L of sorbitol;

step B1-5, mixing and re-suspending the enriched thalli by using an electric transfer culture medium without ions, sucking 1ml of the enriched thalli into a centrifugal tube each time, centrifuging the enriched thalli for 10min at 8500rpm4 ℃ by using a low-temperature refrigerated centrifuge, so that the thalli are gathered at the bottom of the tube, and rinsing the enriched thalli for 4 times by adopting the step, wherein the formula of the electric transfer culture medium without ions is 90g/L of sorbitol, 92.5g/L of mannitol and 100ml/L of glycerol;

and step B1-6, after washing is finished, adding 60 mu L of electrotransformation culture medium into each centrifuge tube by using an aseptic pipette tip to obtain wild type bacillus amyloliquefaciens BA electrotransformation competent cells for later use, wherein the electrotransformation culture medium comprises 90g/L sorbitol, 92.5g/L mannitol and 100ml/L glycerol.

9. The method for preparing genetically engineered bacillus amyloliquefaciens for effectively controlling gray mold as claimed in claim 7, wherein the method comprises the following steps: step B2 includes the following steps:

step B2-1, taking out the freshly prepared wild type Bacillus amyloliquefaciens BA electric transformation competent cells or the competence preserved in the refrigerator from the refrigerator, putting on ice after the competent bacteria liquid is melted, uniformly mixing with 10 mul of recombinant plasmid pHT43/ech42, and carrying out ice water bath for 2 min;

step B2-2, transferring the ice-cooled mixture of step B2-1 to a 1mm electric shock cup, and placing the electric shock cup in a BIO-RADPENE pulser Xcell, and setting five gradients of electric transformation parameters: respectively 12.5kv/cm, 15kv/cm, 17.5kv/cm, 20kv/cm and 21kv/cm, and immediately performing electric shock on the electric shock cup after setting parameters, wherein the electric shock resistance value is 200 omega, and the time constant of the electric shock is 4.5-5.0 ms;

and B2-4, after shaking culture in the step B2-3, centrifuging the centrifuge tube for 1min at 8000g, sucking 900 microliter of supernatant by using a gun head and discarding the supernatant, re-suspending the thallus by using the remaining liquid, coating the thallus in an LB solid culture medium containing chloramphenicol, and culturing in a 37 ℃ constant temperature incubator overnight.

10. Use of genetically engineered bacillus amyloliquefaciens effective in the control of gray mold as claimed in any one of claims 1 to 9, wherein: application in preventing gray mold.