CN107236751B

CN107236751B - Method for rapidly and efficiently screening antibacterial genes by using bacillus subtilis expression system

Info

Publication number: CN107236751B
Application number: CN201710415867.7A
Authority: CN
Inventors: 吴佳; 李梦歌; 苗苗; 董五辈
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2017-06-05
Filing date: 2017-06-05
Publication date: 2019-12-06
Anticipated expiration: 2037-06-05
Also published as: CN107236751A

Abstract

The invention discloses a method for quickly and efficiently screening an antibacterial gene by using a bacillus subtilis expression system, which comprises the following steps: the resistance gene existing in the plant is induced and expressed firstly, a complete cDNA library is constructed, then the cDNA library is introduced into the bacillus subtilis, a secretion expression system of the bacillus subtilis is utilized to express recombinant plasmids, transformants which have lethal effect on the bacillus subtilis are screened firstly, and then nematode-resistant screening and crude protein bacteriostatic screening are further carried out, so that the resistance gene can be obtained quickly and efficiently for subsequent verification work. The invention has the advantages of rapidness, high quality, high efficiency, strong purposiveness, relatively simple screening work and high probability of obtaining positive clones.

Description

method for rapidly and efficiently screening antibacterial genes by using bacillus subtilis expression system

Technical Field

The invention belongs to the field of molecular biology, and relates to a method for quickly and efficiently screening resistance genes by using a bacillus subtilis expression system, which is mainly used for quickly finding the resistance genes and verifying the gene functions.

Background

Bacillus subtilis is an environmentally-friendly (GRAS) gram-positive bacterium certified by the FDA in the united states, and whole genome sequencing has been completed, which has great advantages as an engineered bacterium because: extracellular proteins of bacillus can be directly secreted into a culture medium; the protein secreted by the bacillus is free of endotoxin and is safe; being considered to be very suitable for genetic manipulation and becoming a model organism widely used in laboratory research, bacillus WB800 used in the research has mutated 8 major extracellular proteases, greatly reducing the possibility of degradation of antibacterial peptides. The cDNA library is one of the most commonly used means for studying gene function at present, and is prepared by taking mRNA of specific tissues or cells as a template, carrying out reverse transcription to form cDNA, transferring the cDNA into a proper receptor by using an in vitro DNA recombination technology, and carrying out amplification or expression to form a recombinant clone population.

antimicrobial peptides (AMP) are small molecular polypeptides with broad-spectrum resistance synthesized by organisms, more than 1200 Antimicrobial peptides from animals and plants have been discovered in succession so far, wherein the structures of more than 70 Antimicrobial peptides have been revealed, and the Antimicrobial peptides have a certain killing effect on various fungi, bacteria, parasites and viruses, so that wide attention is paid. Currently, most of the research related to antibacterial peptides focuses on the extraction, separation and purification of active substances and the research on resistance mechanism, and there are three main methods for separating antibacterial peptides: 1. the organism is directly extracted and purified, but the natural synthetic amount of the antibacterial peptide is very small, the loss is large in the separation and purification process, and the inactivation is easy; 2. the method for artificially synthesizing the known antibacterial peptide has high cost and long time consumption, cannot ensure the natural conformation and the biological activity of the antibacterial peptide, and is difficult to screen new antibacterial peptide; 3. the method comprises the steps of constructing an engineering bacteria fusion expression antibacterial gene, utilizing a difference display technology, separating different fragments by means of PCR, screening out a target gene, and after PCR amplification, obviously increasing the repetition rate of the fragments, wherein the separated fragments may be invalid fragments.

According to the invention, a bacillus WB800 secretion system is adopted, firstly, antibacterial peptides are expressed in thalli, so that a screening system becomes more sensitive, secondly, a library screening method is adopted, a plurality of potential antibacterial peptides can be screened, and simultaneously, corresponding gene segments are correspondingly arranged, and finally, bacillus is a biocontrol bacterium, so that the discovered new antibacterial peptides have great application potential.

Disclosure of Invention

the invention aims to provide a method for quickly and efficiently screening antibacterial genes by using a bacillus subtilis expression system, which comprises the steps of firstly inducing the expression of resistance genes in plants, then selecting a proper vector to construct a complete cDNA library, introducing the cDNA library into bacillus subtilis, then expressing recombinant plasmids by using a secretion expression system of the bacillus subtilis, screening the resistance phenotypes by using a spotting method, and is simple and easy in experimental steps, suitable for library screening with large workload, capable of quickly and efficiently obtaining clones with the resistance phenotypes in limited time, and providing a new way for screening the resistance genes.

in order to achieve the purpose, the invention adopts the following technical measures:

A method for rapidly and efficiently screening an antibacterial gene by using a bacillus subtilis expression system comprises the following steps:

1) Preparation of a cDNA library containing the target Gene:

a. Inoculating pathogenic bacteria (the pathogenic bacteria can cause diseases on host plants and have no requirement on the types of the pathogenic bacteria) on healthy plants to induce the expression of resistance genes, and extracting the total RNA of target plants;

b. Separating and purifying mRNA;

c. Designing Oligo dt containing Xba I enzyme cutting site, wherein the sequence is 5'-ACAGGCTCTAGAGCTTTTTTTTTT TTTTTTTTTTTTT-3', synthesizing a first strand of cDNA, and synthesizing a second strand of cDNA by taking the first strand as a template; after the cDNA synthesis is completed, adding a linker containing NdeI restriction enzyme cutting sites, wherein the linker containing NdeI restriction enzyme cutting sites has 3 pairs, so as to reduce the probability of frame shift mutation, and the method comprises the following steps:

mixing NdeI 1 and NdeI 2, NdeI 3 and NdeI 4, NdeI 5 and NdeI 6 in equal amount, adding 1/10 volume of 10 × PCR buffer, heating at 95 ℃ for 5 minutes in a PCR instrument, and naturally cooling to room temperature to form connectors NdeI 1/NdeI 2, NdeI 3/NdeI 4 and NdeI 5/NdeI 6;

The linkers NdeI 1/NdeI 2, NdeI 3/NdeI 4 and NdeI 5/NdeI 6 were mixed in equal amounts and stored at-20 ℃ until use.

Cdna ligation vector: c, performing double digestion on the cDNA added with the linker in the step c by using restriction endonucleases Xba I and Nde I, and connecting the cDNA to a plasmid pBE-S;

e. Transforming the connection system into high-efficiency competent cells of escherichia coli, and amplifying plasmids; after the plasmids are extracted from the population, transforming the WB800 bacillus subtilis, selecting not less than 2000 transformants and storing for later use;

2) Screening for lethal Strain

a. after the transformant is shaken in an LB culture medium marked with kanamycin resistance for one night, the transformant is spotted on a flat plate with the same resistance, a strain which originally grows normally but becomes transparent or semitransparent in the later period is screened out, and the gene has lethal effect through preliminary confirmation;

b. shaking the preliminarily confirmed bacteria liquid containing the lethal gene, extracting plasmids, amplifying escherichia coli, and then transforming the bacillus subtilis WB800 and the bacillus subtilis wild strain 168 again to obtain a lethal phenotype, wherein the lethal phenotype is caused by the inserted gene and is used as a candidate gene of the next step;

3) screening of nematode-resistant genes

The resistance of transformants containing the candidate gene to caenorhabditis elegans is determined by adopting a filter paper method, a selection index is determined by taking bacillus subtilis WB800 as a control, and the selection index formula is as follows: selecting an index (the number of nematodes in the test bacteria-the number of nematodes in the control bacteria)/the total number of the nematodes, and selecting transformants with the index of less than-30% to have the nematode resistance effect for the next screening;

4) Screening of antibacterial genes

Extracting extracellular crude protein of the strain containing the anti-nematode gene in the step 3) by an ammonium sulfate precipitation method, determining the bacteriostatic effect of the crude protein by a filter paper sheet method by taking an empty carrier strain as a reference, and extracting plasmids and gene sequencing to obtain the corresponding antibacterial gene.

Preferably, the ligation system is transformed into escherichia coli high-efficiency competent cells in the step 1), wherein the competent cells are escherichia coli competent HST08, so that the transformation efficiency is improved, and the loss of genes is reduced.

Compared with the prior art, the invention has the following advantages and beneficial effects:

In the lethal screening process, the antibacterial peptide is expressed in the thalli at first and is secreted to the outside of the cell finally, the antibacterial peptide in the body is accumulated and acts, so that a screening system becomes more sensitive, and corresponding gene fragments, nematode-resistant gene screening and crude protein bacteriostatic screening can be carried out while a plurality of potential antibacterial peptides can be screened, and genes with nematode-resistant or antibacterial effects can be screened from a plurality of potential antibacterial peptides. Finally, the bacillus is a biocontrol bacterium, and the discovered novel antibacterial peptide has great potential in the aspect of direct application.

After the antibacterial peptide is found, the gene sequence can be determined through gene sequencing, then whether the antibacterial peptide is a new antibacterial gene or not is preliminarily determined through comparison of various large databases, and if the comparison has no result, the antibacterial peptide sequence can be further compared, and finally whether the antibacterial peptide is the new antibacterial peptide or not is determined.

Drawings

FIG. 1 is a gel electrophoresis test chart of extracted total RNA.

There are 3 lanes, DL2000 DNA marker in lane 1, and total RNA gel detection in lanes 2 and 3. The bands of 18s rRNA and 28s rRNA are clear.

FIG. 2 is a gel electrophoresis image of purified mRNA.

There are 2 lanes, DL2000 DNA marker in lane 1 and mRNA gel detection in lane 2. mRNA is in diffuse bands.

FIG. 3 is a gel electrophoresis test of double-stranded cDNA.

there are 2 lanes, 100bp DNA Ladder in lane 1 and cDNA gel detection in lane 2. The cDNA is a diffuse band.

FIGS. 4 and 5 are the random PCR detection diagrams of the inserts of the Isatis root cDNA library and pinellia ternate cDNA library, respectively.

there are 50 lanes, lane 13 is 100bp DNA Ladder, and the other lanes are randomly selected strains in the library.

FIG. 6 is a diagram showing a lethal pattern of a gene containing a resistance gene.

panel A is a colony morphology map after 12h spotting, and panel B is a colony morphology map after 24h spotting. The left side colony is empty carrier colony in the culture dish, and is the control colony, and the right side colony is lethal strain colony, test fungus colony promptly.

FIG. 7 is a scanning electron micrograph of a lethal bacterium.

FIG. A is a scanning electron microscope image of a WB800 strain of wild type Bacillus subtilis, FIG. B is a scanning electron microscope image of an empty vector strain transferred into a pBE-S vector, and FIG. C is a scanning electron microscope image of a lethal strain.

FIG. 8 is a graph of the bacteriostatic effect of crude protein of test bacteria.

In the figure, the indicator bacteria are wild bacillus subtilis WB800, the white circles are filter paper sheets, the crude protein with the inhibition zone around the filter paper sheets is the crude protein of the test bacteria, and the crude protein without the inhibition zone around the filter paper sheets is the crude protein of the empty carrier strain.

FIG. 9 is a diagram showing the effect of screening nematode-resistant genes by a filter paper sheet method.

the pattern A is the pattern of nematodes and wormholes under the control wild type bacillus subtilis WB800 filter paper, and the pattern B is the pattern of nematodes and wormholes under the test bacteria filter paper.

Detailed Description

Example 1: screening of antibacterial genes in isatis root

1. Preparation of an isatis root cDNA library:

(1) after the radix isatidis is cultured for 4 weeks, the corn sheath blight pathogenic bacteria AGI-IA are inoculated, and the sealing film is sealed for moisture preservation. Inducing the large-scale expression of the resistance gene of the isatis root, sampling once every 6h, immediately storing at ultralow temperature of-70 ℃ for later use after the sampling is finished until the isatis root leaves are attacked, and not sampling any more. Using a cell total RNA extraction reagent Trizol to extract a large amount of total RNA of the isatis leaf, wherein the concentration after enrichment is 2600 ng/mu L. 5 μ L of RNA was subjected to gel electrophoresis, RNA quality was measured (see FIG. 1), and the remainder was stored at-70 ℃ at ultralow temperature until the total volume reached 500 μ L, and then purified.

(2) Magnetic bead method (mRNA Isolation systems), experiments are carried out according to the experimental manual, if the purification concentration is low, a plurality of magnetic beads can be used for enrichment, the concentration measured after the mRNA is purified is 260 ng/mu L, 5 mu L of mRNA is taken for gel electrophoresis, the mRNA quality is detected (see figure 2), and the residual mRNA is immediately subjected to reverse transcription.

(3) Designing Oligo dt containing Xba I restriction site to synthesize the first strand of cDNA, and synthesizing the second strand of cDNA using the first strand as template.

First strand cDNA Synthesis System:

mRNA(5μg)	11μL
		5×1^ststrand synthesis buffer	4μL
dNTP Mixture	1μL
		Rnase inhibitor	1μL
OligodTprimer (containing Xba I cleavage site)	2μL
		RTnase	1μL
Total volume	20μL

the Oligo dt sequence containing the XbaI cleavage site was 5'-ACAGGCTCTAGAGCTTTTTTTTTTTTTTTT TTTTTTT-3' and was used for binding mRNA during mRNA purification. After mRNA was heated at 65 ℃ for 5min, it was immediately taken out of ice and left to stand for 5min (cooling). Adding the above reaction solution, standing at room temperature for 10min, transferring to 42 deg.C constant temperature water bath (which can be made in PCR instrument), and reacting for 1 h. After the reaction, the reaction mixture was cooled for 2min on ice.

second strand cDNA Synthesis System:

The following reaction solution was added to the first strand synthesized microcentrifuge tube, and the total volume was 142. mu.L:

5×2^ststrand synthesis buffer	30μL
		dNTP Mixture	3μL
Rnase-free H₂O	89μL

adding 2 μ L of E.coli DNA polymersel and 2 μ L of E.coli RNaseH/E.coli DNA ligand mixture, mixing gently, reacting at 16 deg.C for 2 h; heating at 70 deg.C for 10min, taking out, and standing at room temperature for 5 min; adding 4 mu L of T4 DNA polymerase, mixing gently, reacting at 37 ℃ for 10 min; adding RNase-free H2O into the second strand cDNA synthesis reaction solution (150. mu.L) to a final volume of 500. mu.L, adding an equal amount of phenol/chloroform/isoamyl alcohol (25:24:1) solution, and shaking by vortex for 5-10 s; centrifuging at 15000rpm for 5min at room temperature, and carefully removing the upper aqueous phase to another new microcentrifuge tube; 1/10 volumes (50. mu.L) of 3M sodium acetate (pH 5.2), 2.5 times the amount of 100% ethanol were added, mixed well and immediately centrifuged at 15000rpm for 30 min; discarding the supernatant, washing with 70% ethanol, and repeating the washing process once; after drying the precipitate, the precipitate was dissolved with 20. mu.L of RNase-free H2O (see FIG. 3 for assay results) at a concentration of 1300 ng/. mu.L and stored at-20 ℃ for subsequent use in adding linkers.

The method for preparing the linker containing NdeI enzyme cutting site comprises the following steps: the synthesized oligonucleotides were dissolved in the corresponding volumes of double distilled water or TE buffer as indicated above in the synthesis protocol to a concentration of 100 pm/. mu.L. NdeI 1 and NdeI 2, NdeI 3 and NdeI 4, NdeI 5 and NdeI 6 were mixed in equal amounts in a PCR tube and 1/10 volumes of 10 XPCR buffer were added. Heated at 95 ℃ for 5 minutes in a PCR apparatus, and then naturally cooled to room temperature (20-25 ℃ C., the same applies hereinafter). The joints formed were as follows:

The linkers NdeI 1/NdeI 2, NdeI 3/NdeI 4 and NdeI 5/NdeI 6 were mixed in equal amounts and stored at-20 ℃ for later use.

adding a linker containing NdeI cleavage site (PrimeScript Double Strand cDNA Synthesis Kit), adding a NdeI linker system:

cDNA	3μL
		10×T₄ DNA ligase buffer	2μL
NdeⅠ adaptor	0.5μL
		T₄ DNA ligase	1μL
ddH₂O	13.5μL
		Total volume	20μL

After mixing gently, the mixture was reacted at 16 ℃ for about 10 hours. Preserving the temperature at 70 ℃ for 30min, standing at room temperature for 5min, and immediately performing double enzyme digestion treatment.

double enzyme digestion vector system:

after mixing gently, the mixture was reacted at 37 ℃ for 3 to 4 hours. And (3) recovering a large fragment in the double enzyme digestion product by using a PCR gel cutting recovery kit, eluting by using 15-20 mu L ddH2O, detecting the concentration by using a spectrophotometer to be 1000 ng/mu L, and converting the recovered product into escherichia coli to detect whether the enzyme digestion is complete.

double restriction digestion of cDNA System:

10×M buffer	4μL
		NdeⅠ	2μL
XbaⅠ	2μL
		cDNA	20μL
ddH₂O	12μL
		Total volume	40μL

After mixing gently, the mixture was reacted at 37 ℃ for 3 to 4 hours. The double digestion products were removed using PCR clean recovery kit, eluted with 15. mu.L-20. mu.L ddH2O, and detected with a spectrophotometer at a concentration of 1000 ng/. mu.L for subsequent ligation of the vector.

(4) cDNA ligation vector: the cDNA added with the linker is connected to a signal peptide library plasmid pBE-S (purchased from Takara Bio-engineering Co., Ltd.) containing 173 signal peptides after double enzyme digestion, the expression and secretion of recombinant proteins are closely related to the types of the signal peptides, and a signal peptide library is connected to a carrier, which is favorable for screening different secreted proteins.

A connection system:

cDNA	12μL
		pBE-S	1μL
10×T₄ DNA ligase buffer	2μL
		T₄ DNA ligase	1μL
ddH₂O	4μL
		Total volume	20μL

The PCR instrument was incubated at 16 ℃ for 3-5h, and this 20. mu.L ligation solution was used for subsequent transformation.

(5) the ligation system is firstly transformed into Escherichia coli high-efficiency competence HST08 (purchased from Wuhan Youth Biotechnology Co., Ltd.), a large amount of amplification recombinant plasmids are coated on a plate, the Bacillus subtilis WB800 strain (purchased from Shanghai Beinoco Biotechnology Co., Ltd.) is transformed after plasmids are extracted from a group, no less than 2000 transformants are selected and stored for later use, so that the subsequent secretion expression of recombinant proteins is carried out.

2. screening for lethal Strain

(1) After the transformant is shaken and cultured in LB culture medium with the final concentration of the marked resistant kanamycin being 10 mug/mL for one night, the transformant is spotted on a plate with the same resistance, the colony morphology is observed periodically, the strain with good growth vigor is found, the strain begins to die slowly after growing for a certain time, the colony is slowly digested from the thick and compact morphology and becomes transparent or semitransparent (see figure 6), the colony morphology of the lethal strain is observed by a scanning electron microscope, and the strain is found to be distorted, deformed or empty in the middle (see figure 7). It was preliminarily confirmed that the gene had lethal effect. We used this method to screen a total of 60 lethal strains and selected strain number 915 for subsequent validation.

(2) After the 915 strain is shake-cultured for 8-12h, a plasmid is extracted, the bacillus subtilis WB800 is transformed again, the lethal phenotype still appears, the plasmid is transformed into a bacillus subtilis wild strain 168 (stored in a laboratory), the lethal phenotype also appears, and the lethal phenomenon does not appear in an empty vector strain. It was further shown that the lethal phenotype is caused by the inserted gene, which plays a lethal role and serves as a candidate gene for the next step.

3. Screening of nematode-resistant genes

A filter paper sheet method: placing 0.22 mu m organic filter membranes on two sides of an NGM plate, respectively placing 915 strains and bacillus subtilis WB800 strains which are cultured for 72 hours on the filter membranes, uncovering escherichia coli OP50 on the filter membrane points after 12 hours of growth, culturing for 12 hours, placing 70-80L 4 C.elegans on the middle point, counting the number of nematodes in colonies on two sides after 10 hours (see figure 9), and obtaining the following data after multiple and mass screening: 263 nematodes are shared at the secretion of 915, 682 nematodes are shared at the secretion of the WB800 strain on the other side, and the selection index is used as an index (the selection index is (number of nematodes in test bacteria-number of nematodes in control bacteria)/total number of nematodes), and-44.34% is used at the selection index of 915 (transformants with the selection index less than-30% have nematode resistance), so that the secretion of the 915 strain can be determined to have certain nematode resistance and can be used for next screening.

4. Screening of antibacterial genes

915 strains are transferred into 50mL of the same resistant LB after being activated for 2 generations on a resistant LB plate with 10 mu g/mL of kanamycin final concentration and are transferred into the same resistant LB for shaking culture for 12h, then the strains are inoculated into 200mL of the same resistant LB according to the inoculation amount of 2 percent and are shaken and fermented for 72h at 37 ℃, 30mL of supernatant is taken and extracted into crude protein by the method of ammonium sulfate precipitation, protein is dissolved by PBS, and salt ions are removed by dialysis. The antibacterial effect of the gene is measured by a filter paper method by taking an empty carrier strain as a contrast, and the 915 strain is found to have a certain antibacterial effect on various bacteria such as bacillus subtilis WB800 (shown in figure 8), the empty carrier strain, bacterial wilt bacteria, bacterial blight bacteria and the like. After plasmid extraction is carried out on 915 bacterial strains, and gene sequences are obtained through sequencing, any result is not compared in each large database, corresponding protein sequences are compared, the result is not compared, and the gene can be determined to be a new antibacterial gene.

Example 2: screening of antibacterial genes in pinellia ternata

1. Preparation of pinellia ternata cDNA library:

Taking out healthy pinellia ternata from a culture pot, washing tubers with sterile water, pricking 7-10 holes on each healthy tuber by using a sterile syringe needle, putting the tubers into the cultivated pinellia ternata soft rot germ liquid, soaking for 15-20min, taking out, re-planting the tubers into the culture pot, culturing in an incubator, inducing a large amount of expression of resistance genes of the pinellia ternata, sampling once every 6h, immediately storing at-70 ℃ ultralow temperature for later use after the tubers are taken out until the pinellia ternata root medium is diseased, and taking out no more samples. The total RNA of pinellia tuber leaves is extracted in large quantity by using a cell total RNA extraction reagent Trizol, and the concentration after enrichment is 9675 ng/mu L. Taking 5 mu L of RNA for gel electrophoresis, detecting the RNA quality, preserving the rest at the ultralow temperature of-70 ℃ for later use until the total volume reaches 500 mu L, and purifying.

(2) separating and purifying mRNA (mRNA Isolation systems) by a magnetic bead method, carrying out experiments according to an experimental manual, if the purification concentration is low, enriching by using a plurality of magnetic beads, obtaining the concentration of 188 ng/mu L after purifying the mRNA, carrying out gel electrophoresis on 5 mu L of the mRNA, detecting the quality of the mRNA, and immediately carrying out reverse transcription on the rest mRNA.

first strand cDNA Synthesis System:

the Oligo dt sequence containing the XbaI cleavage site was 5'-ACAGGCTCTAGAGCTTTTTTTTTTTTTTTTTTTTTTT-3' and was used for binding mRNA during mRNA purification. After mRNA was heated at 65 ℃ for 5min, it was immediately taken out of ice and left to stand for 5min (cooling). Adding the above reaction solution, standing at room temperature for 10min, transferring to 42 deg.C constant temperature water bath (which can be made in PCR instrument), and reacting for 1 h. After the reaction, the reaction mixture was cooled for 2min on ice.

Second strand cDNA Synthesis System:

Adding 2 μ L of E.coli DNA polymersel and 2 μ L of E.coli RNaseH/E.coli DNA ligand mixture, mixing gently, reacting at 16 deg.C for 2 h; heating at 70 deg.C for 10min, taking out, and standing at room temperature for 5 min; adding 4 mu L of T4 DNA polymerase, mixing gently, reacting at 37 ℃ for 10 min; adding RNase-free H2O into the second strand cDNA synthesis reaction solution (150. mu.L) to a final volume of 500. mu.L, adding an equal amount of phenol/chloroform/isoamyl alcohol (25:24:1) solution, and shaking by vortex for 5-10 s; centrifuging at 15000rpm for 5min at room temperature, and carefully removing the upper aqueous phase to another new microcentrifuge tube; 1/10 volumes (50. mu.L) of 3M sodium acetate (pH 5.2), 2.5 times the amount of 100% ethanol were added, mixed well and immediately centrifuged at 15000rpm for 30 min; discarding the supernatant, washing with 70% ethanol, and repeating the washing process once; after drying the precipitate, the precipitate was dissolved with 20. mu.L of RNase-free H2O at a concentration of 567 ng/. mu.L and stored at-20 ℃ for subsequent use in the addition of linkers. The linker containing Nde I cleavage site was prepared as described in example 1.

Adding a linker system containing Nde I enzyme cutting sites:

cDNA	2μL
		10×T₄ DNA ligase buffer	2μL
NdeⅠadaptor	1μL
		T₄ DNA ligase	1μL
ddH₂O	14μL
		total volume	20μL

after mixing gently, the mixture was reacted at 16 ℃ for about 10 hours. Preserving the temperature at 65 ℃ for 30min, standing at room temperature for 5min, and immediately performing double enzyme digestion treatment.

Double restriction digestion of cDNA System:

10×M buffer	4μL
		NdeⅠ	2μL
XbaⅠ	2μL
		Linker-added cDNA	20μL
ddH₂O	12μL
		Total volume	40μL

After mixing gently, the mixture was reacted at 37 ℃ for 3 to 4 hours. The double digestion products were removed using a PCR clean recovery kit, eluted with 40. mu.L ddH2O, and detected with a spectrophotometer at a concentration of 23.5 ng/. mu.L to prepare for subsequent ligation of the vector.

E.coli strains containing empty vectors were shake-cultured and activated, and plasmids were extracted according to the kit method at a concentration of 550 ng/. mu.L.

Double enzyme digestion vector system:

10×green buffer	6μL
		NdeⅠ	3μL
XbaⅠ	3μL
		pBE-S	7μL
ddH₂O	41μL
		Total volume	60μL

After mixing gently, the mixture was reacted at 37 ℃ for 3 to 4 hours. And (3) recovering a large fragment in the double enzyme digestion product by using a PCR gel cutting recovery kit, eluting by using 25 mu L ddH2O, detecting the concentration to be 58 ng/mu L by using a spectrophotometer, and converting the recovered product into escherichia coli to detect whether the enzyme digestion is complete.

(4) cDNA ligation vector: the cDNA added with the linker is connected to a signal peptide library plasmid pBE-S (purchased from Dalianbao Biotechnology Co., Ltd.) containing 173 signal peptides after double enzyme digestion, the expression and secretion of the recombinant protein are closely related to the types of the signal peptides, and a signal peptide library is connected to a carrier, which is favorable for screening different secreted proteins.

A connection system:

cDNA	6.5μL
		pBE-S	6μL
10×T₄ DNA ligase buffer	2μL
		T₄ DNA ligase	1μL
ddH₂O	4.5μL
		Total volume	20μL

The PCR instrument is incubated at 16 ℃ for 3-5h, and the 20. mu.L of the ligation solution can be stored at-20 ℃ for later use in transformation.

(5) The ligation system was transformed into highly competent Escherichia coli HST08, and the recombinant plasmids were amplified in large quantities by plating, and the concentration of the plasmids extracted from the population was 1958 ng/. mu.L. And transforming the Bacillus subtilis WB800 strain, selecting 2039 transformants, and storing for later use so as to perform subsequent secretory expression of the recombinant protein.

2. Screening for lethal Strain

(1) after the transformant is shaken in an LB culture medium with the final concentration of the marker resistant Kanamycin of 10 mug/mL for overnight culture, the sample is applied to a plate with the same resistance, the colony morphology is observed regularly, the strain with good growth vigor is found, the strain begins to die slowly after growing for a certain time, the colony is slowly digested from the thick and compact morphology and becomes transparent or semitransparent, the scanning electron microscope is used for observing the colony morphology of the lethal strain, and the strain is found to be deformed or empty in the middle. It was preliminarily confirmed that the gene had lethal effect. We used this method to screen for 95 lethal strains in total, and selected strain number 1366 for subsequent validation.

(2) After the 1366 strain is shake-cultured for 8-12h, a plasmid is extracted, the bacillus subtilis WB800 is transformed again, the lethal phenotype still appears, the plasmid is transformed into the bacillus subtilis wild strain 168, the lethal phenotype also appears, and the lethal phenomenon does not appear in the empty vector strain. It was further shown that the lethal phenotype is caused by the inserted gene, which plays a lethal role and serves as a candidate gene for the next step.

3. screening of nematode-resistant genes

A filter paper sheet method: placing 0.22 mu m organic filter membranes on two sides of an NGM plate, respectively placing 1366 strains and bacillus subtilis WB800 strains which are cultured for 72 hours on the filter membranes, after 12 hours of growth, uncovering escherichia coli OP50 on the filter membrane points, after 12 hours of culture, placing 70-80L 4 S.elegans on the middle point, counting the number of nematodes in bacterial colonies on two sides after 10 hours, and obtaining the following data after multiple times of mass screening: 644 nematodes are shared in the 1366 secretion, 1369 nematodes are shared in the WB800 strain secretion on the other side, and the 1366 selection index is-36.02% (the selection index is-30% of transformants with nematode resistance) by using the selection index as an index (the selection index is equal to (number of nematodes in test bacteria-number of nematodes in control bacteria)/total number of nematodes), so that the 1366 strain secretion can be determined to have certain nematode resistance for the next screening.

4. Screening of antibacterial genes

The 1366 strain was activated on a resistant LB plate containing 10. mu.g/mL kanamycin to a final concentration of 10. mu.g/mL for 2 generations, transferred to 50mL of the same resistant LB for 12h by shaking, inoculated to 200mL of the same resistant LB at a 2% inoculum size, fermented at 37 ℃ for 72h by shaking, 30mL of the supernatant was precipitated with ammonium sulfate to extract crude protein, the protein was solubilized with PBS, and the salt ions were removed by dialysis. The antibacterial effect of the gene is measured by a filter paper method by taking an empty carrier strain as a contrast, and the 1366 strain is found to have a certain antibacterial effect on WB800, the empty carrier strain, bacterial wilt bacteria, bacterial leaf blight bacteria and other bacteria. After plasmid extraction is carried out on the 1366 strain, and the sequence is sequenced and the gene sequence is taken, any result is not compared in each large database, the corresponding protein sequence is compared, and the result is not compared, so that the gene can be determined to be a new antibacterial gene.

the above list is only 2 specific examples of the present invention, and the experimental material of the present invention may be other plants, for example, edible crops such as garlic and corn, medicinal plants such as honeysuckle and houttuynia cordata, and biocontrol bacteria such as trichoderma harzianum. The indicator bacteria selected in the antibacterial gene screening can also be various dead body nutritional pathogenic bacteria such as fruit spot pathogenic bacteria, ulcer pathogenic bacteria, rice bacterial streak pathogenic bacteria and the like.

Claims

1. A method for rapidly and efficiently screening an antibacterial gene by using a bacillus subtilis expression system comprises the following steps:

1) Preparation of a cDNA library containing the target Gene:

a. Inoculating pathogenic bacteria on healthy plants, inducing the expression of resistance genes, and extracting total RNA of target plants;

b. separating and purifying mRNA;

c. Designing Oligo dt containing Xba I enzyme cutting site, wherein the sequence is 5'-ACAGGCTCTAGAGCTTTTTTTTTTTTTTTTTTTTTTT-3', synthesizing a first strand of cDNA, and synthesizing a second strand of cDNA by taking the first strand as a template; after the cDNA synthesis is completed, a linker containing NdeI restriction enzyme cutting sites is added, the linker has 3 pairs and the sequence is as follows:

2) Screening for lethal Strain

3) Screening of nematode-resistant genes

The resistance of transformants containing the candidate gene to caenorhabditis elegans is determined by adopting a filter paper method, a selection index is determined by taking bacillus subtilis WB800 as a control, and the selection index formula is as follows: selecting an index (the number of nematodes in a colony of a transformant containing the candidate gene-the number of nematodes in a colony of the bacillus subtilis WB 800)/the total number of nematodes, and selecting transformants with the index of less than-30% to have the nematode resistance effect for the next screening;

4) Screening of antibacterial genes

2. The method for rapidly and efficiently screening antibacterial genes by using a bacillus subtilis expression system according to claim 1, wherein the method comprises the following steps: and step 1), transforming the connection system into an escherichia coli high-efficiency competent cell, wherein the competent cell is escherichia coli competent HST 08.