CN107236751A - A kind of utilization bacillus subtilis expression system is quick, the method for high frequency zone antifungal genes - Google Patents
A kind of utilization bacillus subtilis expression system is quick, the method for high frequency zone antifungal genes Download PDFInfo
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Abstract
The invention discloses a kind of method that utilization bacillus subtilis expression system fast and efficiently screens antifungal genes, its step is：Resistant gene present in first induced expression plant, build a complete cDNA library, it imported into again in bacillus subtilis, utilize its secreting, expressing system, recombinant expression, the transformant that producing bacillus subtilis are given birth to lethal effect is first screened, further by the screening of anti-nematode and the antibacterial screening of crude protein, subsequent authentication work is carried out so as to the resistant gene that obtains rapidly and efficiently.Of the invention quick, high-quality, efficiently, and purpose is strong, screening operation is relatively easy, and the probability for obtaining positive colony is high.
Description
Technical field
The invention belongs to biology field, it is related to a kind of using bacillus subtilis expression system is quick, high-efficiency sieve
The method for selecting resistant gene, it is mainly used in being quickly found out resistant gene, and is able to verify that gene function.
Background technology
Bacillus subtilis is a kind of (GRAS) gram-positive bacterium of environmental sound through U.S. FDA certification, entirely
Gene order-checking has been completed, and it has very big advantage as engineering bacteria, and reason is：The extracellular protein of bacillus can be with
Directly it is secreted into culture medium；The albumen endotoxin-free of secreted from bacillus, it is safer；It is considered as being especially suitable for hereditary behaviour
Make, and turn into the model organism for being widely used in laboratory research, bacillus WB800 used, has dashed forward in this research
Become and fallen 8 main extracellular proteases, greatly reduce the possibility of antibacterial peptide degraded.CDNA library is specifically to organize
Or the mRNA of cell is template, the cDNA of reverse transcription formation recycles external DNA recombinant techniques, is transferred to suitable acceptor and enters
Row amplification is expressed, and forms the clonal population of a restructuring, cDNA library be the most frequently used means of current research gene function it
One.
Antibacterial peptide (antimicrobial peptide, AMP) is many by the small molecule with resistance of wide spectrum of biosynthesis
Peptide, up to the present more than the 1200 kinds of antibacterial peptide from animals and plants is found successively, wherein the knot of more than 70 kinds of antibacterial polypeptide
Structure has been revealed, and antibacterial peptide has certain lethal effect to cause widely a variety of fungies, bacterium, parasite and virus
Pay attention to.In terms of the correlative study of current antibacterial peptide has focused largely on the extraction separation and purifying of active material and resistance mechanism
In research, the method for separation antibacterial peptide mainly has three kinds：1st, the direct extraction purification of organism, but the natural synthetic quantity of antibacterial peptide is non-
It is often few, lose big during isolating and purifying, and easily inactivation；2nd, artificial synthesized known antibacterial peptide, the method cost is high, time-consuming
It is long, it is impossible to ensure the native conformation and bioactivity of antibacterial peptide, and be difficult to screen new antibacterial peptide；3rd, engineering bacteria is built to melt
Expression antifungal genes are closed, and this method utilizes differential display technique, discrepant fragment separated by PCR means, screens
Go out target gene, after being expanded through PCR, the repetitive rate of fragment is dramatically increased, and the fragment of separation may be invalid fragments, in addition,
Another method is the separation detection step by step based on albumen or polypeptide, and the gene order being inferred to of starting with from activated protein is synthesized again
Antibacterial peptide, the method work period is long, and workload is big, and the antibacterial peptide synthesized again often it is not anticipated that activity.
The present invention uses bacillus WB800 excretory systems, and antibacterial peptide is in thalline internal representations first so that screening system
Become sensitiveer, secondly, can be while being to filter out many potential antibacterial peptides using the method for library screening, correspondence
Upper corresponding gene fragment, finally, inherently one biocontrol microorganisms of bacillus, the new antibacterial peptide of discovery have very big application
Potentiality.
The content of the invention
It is an object of the invention to provide one kind using bacillus subtilis expression system is quick, high frequency zone antifungal genes
Method, this method induces the expression of resistant gene in plant, one complete cDNA of the suitable vector construction of reselection first
Library, imported into after bacillus subtilis, using its secreting, expressing system expression recombinant plasmid, and point sample method screens resistant phenotype,
Experimental procedure is simple, easy, is adapted to the big library screening of workload, can within the limited time being had rapidly and efficiently
The clone of resistant phenotype, new approach has been put forward for screening resistant gene.
In order to realize above-mentioned purpose, the present invention uses following technical measures：
A kind of utilization bacillus subtilis expression system is quick, the method for high frequency zone antifungal genes, and its step is：
1) preparation of the cDNA library containing target gene：
A. pathogen being inoculated with healthy plant, (pathogen be able to must cause a disease on host plant, to pathogen species
Class no requirement (NR)) induction of resistance gene expression after, extract target plant total serum IgE；
B. mRNA is isolated and purified；
C. the Oligo dt of the restriction enzyme site containing Xba I are designed, its sequence is 5 '-ACAGGCTCTAGAGCTTTTTTTTTT
TTTTTTTTTTTTT-3 ', synthesizes first chain of cDNA, using first chain as templated synthesis Article 2 cDNA chains；CDNA has been synthesized
Cheng Hou, adds the joint of the restriction enzyme site containing Nde I, the joint of the restriction enzyme site containing Nde I has 3 pairs, to reduce frameshit
The probability of mutation, is made by following methods：
By Nde I 1 and Nde I 2, Nde I 3 and Nde I 4, Nde I 5 and the mixed in equal amounts of Nde I 6, and add 1/10 volume 10 ×
PCR Buffer, 95 DEG C are heated 5 minutes in PCR instrument, then naturally cool to room temperature, form joint Nde I 1/Nde I 2, Nde I
The 5/Nde I 6 of 3/Nde I 4, Nde I；
By the 3/Nde I 4 of joint Nde I 1/Nde I 2, Nde I and the mixed in equal amounts of I 5/Nde of Nde I 6, -20 DEG C are stored in, it is standby.
D.cDNA connection carriers：By the cDNA for adding joint the restriction endonuclease Xba I and Nde I in step c
After double digestion, it is connected on plasmid pBE-S；
E. linked system is transformed into efficient competent cell of escherichia coli, expands plasmid；Converted after colony's upgrading grain
Bacillus subtilis WB800, no less than 2000 transformants of picking, is saved backup；
2) screening of lethal bacterial strain
A. transformant is shaken in the LB culture mediums of mark kalamycin resistance after training overnight, on the flat board of identical resistance
Point sample, filters out script growing way normally but the later stage becomes the bacterial strain of transparent or semitransparent shape, preliminary to confirm, the gene has lethal
Effect；
B. plasmid is extracted after the above-mentioned bacterium solution containing lethal gene tentatively confirmed being shaken into training, is turned again after being expanded through Escherichia coli
Change bacillus subtilis WB800 and bacillus subtilis wild strain 168, occur in that lethal phenotype, show lethal phenotype by
The gene of insertion causes, the gene as next step candidate gene；
3) screening of nematode resistance gene
Resistance of the transformant containing candidate gene to Caenorhabditis elegans is determined using filter paper enzyme, with bacillus subtilis
WB800 determines selection index as control, and selection exponential formula is as follows：Select index=(nematode population-control in test bacterium
Nematode population in bacterium)/line insect number, select transformant of the index less than -30% that there is anti-nematode to act on, the sieve for next step
Choosing；
4) screening of antifungal genes
Ammonium sulfate precipitation method extraction step 3) in the bacterial strain containing nematode resistance gene extracellular crude protein, using empty carrier bacterial strain as
Control, filter paper enzyme determines the fungistatic effect of crude protein, extracting plasmid and gene sequencing, thus obtains corresponding antifungal genes.
Preferably, step 1) in linked system is transformed into efficient competent cell of escherichia coli, described competence
Cell is E. coli competent HST08, improves transformation efficiency, subtracts oligogenic loss.
Compared with prior art, the present invention has advantages below and beneficial effect：
Lethal screening process allows antibacterial peptide in thalline internal representations first in the present invention, be finally secreted into it is extracellular, it is anti-in vivo
Bacterium peptide is accumulated and worked so that screening system becomes sensitiveer, and can filter out the same of many individual potential antibacterial peptides
When, corresponding gene fragment in correspondence, nematode resistance gene screening and the antibacterial screening of crude protein can be in numerous potential antibacterial peptides
In, filter out the gene with anti-nematode or antibacterial action.Finally, inherently one biocontrol microorganisms of bacillus, discovery it is new
Antibacterial peptide there are very big potentiality in direct application aspect.
It was found that after antibacterial peptide, gene sequencing can be first passed through and determine gene order, then by the comparison of each large database concept come
Primarily determine that whether be new antifungal genes, if compared without result, can further compare antibacterial peptide sequence, it is final to determine
Whether it is new antibacterial peptide.
Brief description of the drawings
Fig. 1 is the total serum IgE detected through gel electrophoresis figure extracted.
3 swimming lanes are had, 1 swimming lane is DL2000DNA maker, and 2,3 swimming lanes are total serum IgE gel detection result.18s
RRNA, 28s rRNA bands are clear.
Fig. 2 is mRNA detected through gel electrophoresis figures after purification.
2 swimming lanes are had, 1 swimming lane is DL2000DNA maker, and 2 swimming lanes are m RNA gel detection results.MRNA is more
Bulk band.
Fig. 3 is double-strand cDNA detected through gel electrophoresis figures.
2 swimming lanes are had, 1 swimming lane is 100bp DNA Ladder, and 2 swimming lanes are cDNA gel detection results.CDNA is more
Bulk band.
Fig. 4, Fig. 5 are respectively Radix Isatidis cDNA library and tuber of pinellia cDNA library Insert Fragment random PCR detection figure.
Totally 50 swimming lanes, 13 swimming lanes are 100bp DNA Ladder, and other swimming lanes are the bacterial strain in the library selected at random.
Fig. 6 is the figure of dead versions containing resistant gene.
It is colonial morphology figure after point sample 12h to scheme A, and figure B is colonial morphology figure after point sample 24h.Left side bacterium colony is in culture dish
Empty carrier bacterium colony, that is, compare bacterium colony, and right side bacterium colony is lethal bacterial strain bacterium colony, that is, tests bacterium bacterium colony.
Fig. 7 is lethal bacterium scanning electron microscopic picture.
Figure A is wild-type B. subtilis WB800 bacterial strain scanning electron microscope (SEM) photographs, and figure B is the empty carrier for being transferred to pBE-S carriers
Bacterial strain scanning electron microscope (SEM) photograph, figure C is lethal bacteria strain scanning electron microscope (SEM) photograph.
Fig. 8 is test bacterium crude protein fungistatic effect figure.
Indicator bacteria is wild-type B. subtilis WB800 in figure, and white circle is to press down around filter paper, filter paper
The crude protein of bacterium circle is the crude protein of inhibition zone do not occur around test bacterium crude protein, filter paper for the thick egg of empty carrier bacterial strain
In vain.
Fig. 9 is that filter paper enzyme screens nematode resistance gene design sketch.
It is nematode and gallery distribution map under control wild-type B. subtilis WB800 filter papers to scheme A, and figure B is test bacterium
Nematode and gallery distribution map under filter paper.
Embodiment
Embodiment 1：The screening of antifungal genes in Radix Isatidis
1. the preparation of Radix Isatidis cDNA library：
(1) Radix Isatidis was cultivated to after 4 weeks, is inoculated with maize sheath blight pathogen AGI-IA, sealed membrane sealing, moisturizing.Induction
The great expression of Radix Isatidis resistant gene, it is every 6h samplings once, standby in -70 DEG C of cryopreservations immediately after taking, until
Radix Isatidis blade is fallen ill, and is no longer sampled.It is a large amount of to extract Radix Isatidis blade total serum IgE, richness with cell total rna extracts reagent Trizol
Concentration is 2600ng/ μ L after integrating.5 μ L RNA gel electrophoresises are taken, detection RNA mass (see Fig. 1) is remaining in -70 DEG C of ultralow temperature
Save backup, until cumulative volume reaches 500 μ L, purified.
(2) paramagnetic particle method isolate and purify mRNA (Isolation Systems), according to laboratory manual
Tested, if purifying concentration is low, multiple enrichment with magnetic bead can be used, be purified into after mRNA and survey concentration for 260ng/ μ L, take 5 μ L
MRNA gel electrophoresises, detection mRNA mass (see Fig. 2), remaining mRNA reverse transcriptions immediately.
(3) Oligo dt synthesis first chain of cDNA of design restriction enzyme site containing Xba I, using first chain as templated synthesis
CDNA Article 2 chains.
First chain synthetic system of cDNA：
| mRNA(5μg) | 11μL |
| 5×1ststrand synthesis buffer | 4μL |
| dNTP Mixture | 1μL |
| Rnase inhibitor | 1μL |
| Oligo dT primer (restriction enzyme site containing Xba I) | 2μL |
| RTnase | 1μL |
| Cumulative volume | 20μL |
The Oligo dt of the restriction enzyme site containing Xba I sequence is 5 '-ACAGGCTCTAGAGCTTTTTTTTTTTTTTTT
TTTTTTT-3 ', for combining mRNA in mRNA purge processes.After 65 DEG C of heating 5min of mRNA, take out in ice place immediately
5min (cooling).Add above-mentioned reaction solution room temperature and place 10min, being transferred to (can be in PCR instrument in 42 DEG C of constant temperature water baths
Do) reaction 1h.Reaction is placed in ice after terminating and cools down 2min.
CDNA Article 2 chain synthetic systems：
Following reaction solution, the μ L of full dose 142 are added in microcentrifugal tube after first chain synthesis：
| 5×2ststrand synthesis buffer | 30μL |
| dNTP Mixture | 3μL |
| Rnase-free H2O | 89μL |
Add 2 μ L E.Coli DNA Polymerasel and 2 μ L E.Coli RNaseH/E.Coli DNA ligase
Mixture, is gently mixed, 16 DEG C of reaction 2h；70 DEG C of heating 10min, take out room temperature and place 5min；Add 4 μ L T4DNA
Polymerase, is gently mixed, 37 DEG C of reaction 10min；Rnase- is added into Article 2 chain cDNA synthesis reaction solutions (150 μ L)
free H2O adds phenol/chloroform/isoamyl alcohol (25 of equivalent to the μ L of final volume 500:24:1) solution, vortex concussions 5-
10s；15000rpm centrifuges 5min at room temperature, and careful upper strata aqueous phase of taking out is into another new microcentrifugal tube；Add 1/10
The 3M sodium acetates (pH5.2) of volume (50 μ L), 100% ethanol of 2.5 times of amounts, after uniform mixing, immediately under 15000rpm from
Heart 30min；Supernatant is abandoned, is cleaned, can be repeated once with 70% ethanol；After drying precipitated, with 20 μ L RNase-free H2O is molten
Solution precipitation (testing result is shown in Fig. 3), concentration is 1300ng/ μ L, in -20 DEG C of preservations, is easy to the head use of adjunction below.
The method for preparing the joint of the restriction enzyme site containing Nde I：The oligonucleotides of synthesis is dissolved according to the explanation on synthesis list
In the distilled water or TE buffer of respective volume, it is 100pm/ μ L to make concentration.Respectively by Nde I 1 and Nde I 2 in PCR pipe,
Nde I 3 and Nde I 4, Nde I 5 and the mixed in equal amounts of Nde I 6, and add 10 × PCR Buffer of 1/10 volume.95 in PCR instrument
DEG C heating 5 minutes, then naturally cool to room temperature (20-25 DEG C, same as below).The joint of formation is as follows：
Again by the 3/Nde I 4 of joint Nde I 1/Nde I 2, Nde I and the mixed in equal amounts of I 5/Nde of Nde I 6, -20 DEG C are stored in, in case
After use.
Plus joint (the PrimeScriptTMDouble Strand cDNA Synthesis of the restriction enzyme site containing Nde I
), plus the joint systems of Nde I Kit：
| cDNA | 3μL |
| 10×T4DNA ligase buffer | 2μL |
| NdeⅠadaptor | 0.5μL |
| T4DNA ligase | 1μL |
| dd H2O | 13.5μL |
| Cumulative volume | 20μL |
After gently mixing, 16 DEG C are reacted 10h or so.70 DEG C of insulation 30min, room temperature is placed after 5min, immediately at double digestion
Reason.
Double digestion carrier system：
After gently mixing, 37 DEG C of reaction 3-4h.The sheet in double digestion product is reclaimed using PCR gel extractions kit
Section, with 15 μ L-20 μ L ddH2O is eluted, and is 1000ng/ μ L, recovery product conversion large intestine bar with spectrophotometer detectable concentration
Bacterium detects whether that digestion is complete.
Double digestion cDNA systems：
| 10×M buffer | 4μL |
| NdeⅠ | 2μL |
| XbaⅠ | 2μL |
| cDNA | 20μL |
| dd H2O | 12μL |
| Cumulative volume | 20μL |
After gently mixing, 37 DEG C of reaction 3-4h.Double digestion product is removed using PCR cleaning QIAquick Gel Extraction Kits, with 15 μ L-20
μ L dd H2O is eluted, and is 1000ng/ μ L with spectrophotometer detectable concentration, in case connecting carrier below.
(4) cDNA connections carrier：The signal peptide storehouse matter containing 173 kinds of signal peptides will be connected to after the cDNA double digestions for adding joint
On grain pBE-S (purchased from precious bioengineering Co., Ltd), the expression secretion of recombinant protein and the species of signal peptide are closely related,
A signal peptide storehouse is connected on carrier, is conducive to screening different secretory proteins.
Linked system：
| cDNA | 12μL |
| pBE-S | 1μL |
| 10xT4DNAligase buffer | 2μL |
| T4DNAligase | 1μL |
| ddH2O | 4μL |
| Cumulative volume | 20μL |
16 DEG C of insulation 3-5h in PCR instrument, this 20 μ L connection solution can do later transformations use.
(5) linked system is first transformed into Escherichia coli Efficiency Competent HST08 (limited purchased from the friendly name bioengineering in Wuhan
Company) in, apply after flat board largely expands recombinant plasmid, colony's upgrading grain and convert bacillus subtilis WB800 bacterial strains again (purchased from upper
Hai Beinuo bio tech ltd), no less than 2000 transformants of picking picking are saved backup, to carry out subsequent recombination
The secreting, expressing of albumen.
2. the screening of lethal bacterial strain
(1) transformant is shaken in the mark final concentration of 10 μ g/mL of resistance kanamycins LB culture mediums after training overnight, in
Point sample on the flat board of identical resistance, routine observation colonial morphology finds the good bacterial strain of script growing way, is growing into certain time
After start slowly to die, bacterium colony is slowly cleared up from abundant fine and close form, becomes transparent or semitransparent shape (see Fig. 6), scanning electricity
The lethal bacterial strain colonial morphology of sem observation, finds the bacterial strain thalline twist distortion or middle emptying (see Fig. 7).It is preliminary to confirm, the base
Because having lethal effect.We screen 60 lethal bacterial strains with this method altogether, choose the bacterial strain that numbering is 915 and carry out subsequently
Checking.
(2) plasmid is extracted after 915 bacterial strain being shaken into training 8-12h, bacillus subtilis WB800 is converted again, is still caused
Dead phenotype, the plasmid is converted in bacillus subtilis wild strain 168 (being preserved by this laboratory), lethal table is also occurred in that
Type, and empty carrier bacterial strain occurs without lethality.Further demonstrate that the gene that lethal phenotype is caused by the gene inserted, that is, is inserted
Rise lethal effect, the gene as next step candidate gene.
3. the screening of nematode resistance gene
Filter paper enzyme：0.22 μm of organic filter membrane is put in NGM flat boards both sides, 915 bacterial strains and withered grass after training 72h will be shaken
Bacillus WB800 bacterial strains are respectively placed on filter membrane, after growth 12h, are opened after Escherichia coli OP50 on filter membrane point, culture 12h,
L4Stage Caenorhabditis elegans 70-80 only, after 10h counts the nematode population (see Fig. 9) in the bacterium colony of both sides, warp on intermediate point
Repeatedly, after a large amount of screenings, the data drawn are as follows：Nematode 263, opposite side WB800 strain secretes things are had at 915 secretion
Place has 682, to select index as index (selection index=(nematode population in nematode population-control bacterium in test bacterium)/line
Worm sum), 915 selection indexes are -44.34% (there is the transformant that selection index is less than -30% anti-nematode to act on), can be true
Fixed 915 strain secretes things have certain anti-nematode effect, the screening for next step.
4. the screening of antifungal genes
By 915 bacterial strains containing kanamycins, activation 2 is instead of transferred to 50mL afterwards on final concentration of 10 μ g/ml resistance LB flat boards
Shaken in identical resistance LB training 12h, then by 2% inoculum concentration be seeded in the identical resistance LB of 200mL in shaken in 37 DEG C training fermentation 72h,
Take the method for 30mL supernatant ammonium sulfate precipitations to extract crude protein, use PBS soluble proteins, dialysis removes salt ion.With empty carrier
Bacterial strain is control, and filter paper enzyme determines the fungistatic effect of the gene, finds 915 bacterial strains to bacillus subtilis WB800 (see figure
8), the various bacteria such as empty carrier bacterial strain, bacterial wilt opportunistic pathogen, bacterial leaf-blight opportunistic pathogen has certain fungistatic effect.915 bacterial strains are entered
Row plasmid extraction, and be sequenced take after gene order, in each large database concept compare arrive any result, by corresponding albumen
Sequence is compared, and does not also compare result, it may be determined that the gene is new antifungal genes.
Embodiment 2：The screening of antifungal genes in the tuber of pinellia
1. the preparation of tuber of pinellia cDNA library：
By health the tuber of pinellia taken out from culturing pot, stem tuber with aseptic water washing it is clean after, with sterile syringe needle
7-10 hole is pricked on each healthy stem tuber, is put into after soaking 15-20min in the tuber of pinellia Bacteria erwinia bacterium solution shaken and trained and takes
Go out, be planted to again in culturing pot, cultivated in incubator, induce the great expression of tuber of pinellia resistant gene, every 6h samplings once,
It is standby in -70 DEG C of cryopreservations immediately after taking, until the morbidity of tuber of pinellia foundation, is no longer sampled.Use cell total rna extracts reagent
Trizol, a large amount of to extract leaf blade of Pinellia total serum IgE, concentration is 9675ng/ μ L after enrichment.5 μ L RNA are taken to carry out gel electrophoresis, detection
RNA mass, it is remaining standby in -70 DEG C of cryopreservations, until cumulative volume reaches 500 μ L, purified.
(2) paramagnetic particle method isolate and purify mRNA (Isolation Systems), according to laboratory manual
Tested, if purifying concentration is low, multiple enrichment with magnetic bead can be used, be purified into after mRNA and survey concentration for 188ng/ μ L, take 5 μ L
MRNA gel electrophoresises, detect mRNA mass, remaining mRNA reverse transcriptions immediately.
(3) Oligo dt synthesis first chain of cDNA of design restriction enzyme site containing Xba I, using first chain as templated synthesis
CDNA Article 2 chains.
First chain synthetic system of cDNA：
The Oligo dt of the restriction enzyme site containing Xba I sequence is 5 '-ACAGGCTCTAGAGCTTTTTTTTTTTTTTTT
TTTTTTT-3 ', for combining mRNA in mRNA purge processes.After 65 DEG C of heating 5min of mRNA, take out in ice place immediately
5min (cooling).Add above-mentioned reaction solution room temperature and place 10min, being transferred to (can be in PCR instrument in 42 DEG C of constant temperature water baths
Do) reaction 1h.Reaction is placed in ice after terminating and cools down 2min.
CDNA Article 2 chain synthetic systems：
Following reaction solution, the μ L of full dose 142 are added in microcentrifugal tube after first chain synthesis：
Add 2 μ L E.Coli DNA Polymerasel and 2 μ L E.Coli RNaseH/E.Coli DNA ligase
Mixture, is gently mixed, 16 DEG C of reaction 2h；70 DEG C of heating 10min, take out room temperature and place 5min；Add 4 μ L T4DNA
Polymerase, is gently mixed, 37 DEG C of reaction 10min；Rnase- is added into Article 2 chain cDNA synthesis reaction solutions (150 μ L)
free H2O adds phenol/chloroform/isoamyl alcohol (25 of equivalent to the μ L of final volume 500:24:1) solution, vortex concussions 5-
10s；15000rpm centrifuges 5min at room temperature, and careful upper strata aqueous phase of taking out is into another new microcentrifugal tube；Add 1/10
The 3M sodium acetates (ph5.2) of volume (50 μ L), 100% ethanol of 2.5 times of amounts, after uniform mixing, immediately under 15000rpm from
Heart 30min；Supernatant is abandoned, is cleaned, can be repeated once with 70% ethanol；After drying precipitated, the RNase-free H2O with 20 μ L are molten
Solution precipitation, concentration is 567ng/ μ L, in -20 DEG C of preservations, is easy to the head use of adjunction below.The preparation of the joint of the restriction enzyme site containing Nde I
Method is referring to embodiment 1.
Plus the joint system of the restriction enzyme site containing Nde I：
| cDNA | 2μL |
| 10×T4DNA ligase buffer | 2μL |
| NdeⅠadaptor | 1μL |
| T4DNA ligase | 1μL |
| dd H2O | 14μL |
| Cumulative volume | 20μL |
After gently mixing, 16 DEG C are reacted 10h or so.65 DEG C of insulation 30min, room temperature is placed after 5min, immediately at double digestion
Reason.
Double digestion cDNA systems：
| 10×M buffer | 4μL |
| NdeⅠ | 2μL |
| XbaⅠ | 2μL |
| Added joint cDNA | 20μL |
| dd H2O | 12μL |
| Cumulative volume | 20μL |
After gently mixing, 37 DEG C of reaction 3-4h.Double digestion product is removed using PCR cleaning QIAquick Gel Extraction Kits, with 40 μ L's
ddH2O is eluted, and is 23.5ng/ μ L with spectrophotometer detectable concentration, in case connecting carrier below.
Coli strain of the training activation containing empty carrier is shaken, plasmid is extracted according to kit method, concentration is 550ng/ μ L.
Double digestion carrier system：
| 10×green buffer | 6μL |
| NdeⅠ | 3μL |
| XbaⅠ | 3μL |
| pBE-S | 7μL |
| dd H2O | 41μL |
| Cumulative volume | 60μL |
After gently mixing, 37 DEG C of reaction 3-4h.The sheet in double digestion product is reclaimed using PCR gel extractions kit
Section, with 25 μ L ddH2O is eluted, and is 58ng/ μ L with spectrophotometer detectable concentration, and recovery product conversion E. coli detection is
No digestion is complete.
(4) cDNA connections carrier：The signal peptide storehouse matter containing 173 kinds of signal peptides will be connected to after the cDNA double digestions for adding joint
On grain pBE-S (purchased from the precious bio tech ltd in Dalian), the expression secretion of recombinant protein and the close phase of species of signal peptide
Close, a signal peptide storehouse is connected on carrier, be conducive to screening different secretory proteins.
Linked system：
| cDNA | 6.5μL |
| pBE-S | 6μL |
| 10×T4DNA ligase buffer | 2μL |
| T4DNA ligase | 1μL |
| ddH2O | 4.5μL |
| Cumulative volume | 20μL |
16 DEG C of insulation 3-5h in PCR instrument, this 20 μ L connections solution can be with -20 DEG C of preservations, so that later transformations are used.
(5) linked system is first transformed into Escherichia coli Efficiency Competent HST08, applies flat board and largely expand restructuring matter
Grain, colony's upgrading grain, concentration is 1958ng/ μ L.Bacillus subtilis WB800 bacterial strains are converted again, and 2039 transformants of picking are protected
Deposit standby, to carry out the secreting, expressing of subsequent recombination albumen.
2. the screening of lethal bacterial strain
(1) transformant is shaken in the mark final concentration of 10 μ g/mL of resistance Kanamycin LB culture mediums after training overnight,
In point sample on the flat board of identical resistance, routine observation colonial morphology finds the good bacterial strain of script growing way, is growing into a timing
Between after start slowly to die, bacterium colony is slowly cleared up from abundant fine and close form, becomes transparent or semitransparent shape, scanning electron microscopic observation
Lethal bacterial strain colonial morphology, finds the bacterial strain, thalline twist distortion or middle emptying.Preliminary to confirm, the gene has lethal work
With.We screen 95 lethal bacterial strains with this method altogether, choose the bacterial strain that numbering is 1366 and carry out subsequent authentication.
(2) plasmid is extracted after 1366 bacterial strains being shaken into training 8-12h, bacillus subtilis WB800 is converted again, is still occurred lethal
Phenotype, the plasmid is converted in bacillus subtilis wild strain 168, lethal phenotype is also occurred in that, and empty carrier bacterial strain does not go out
Existing lethality.Further demonstrate that lethal phenotype is caused by the gene inserted, that is, the gene inserted plays lethal effect, the gene is made
For the candidate gene of next step.
3. the screening of nematode resistance gene
Filter paper enzyme：Put 0.22 μm of organic filter membrane in NGM flat boards both sides, will shake training 72h after 1366 bacterial strains with it is withered
Careless bacillus WB800 bacterial strains are respectively placed on filter membrane, after growth 12h, are opened Escherichia coli OP50 on filter membrane point, are cultivated 12h
Afterwards, on intermediate point L4Stage Caenorhabditis elegans 70-80 only, after 10h count both sides bacterium colony in nematode population, through repeatedly,
After a large amount of screenings, the data drawn are as follows：Have at nematode 644, opposite side WB800 strain secretes things and be total at 1366 secretion
There are 1369, to select index as index (selection index=(nematode population in nematode population-control bacterium in test bacterium)/nematode
Sum), 1366 selection indexes are -36.02% (there is the transformant that selection index is less than -30% anti-nematode to act on), can be true
Fixed 1366 strain secretes things have certain anti-nematode effect, for next step screening.
4. the screening of antifungal genes
By 1366 bacterial strains containing kanamycins, activation 2 is instead of transferred to afterwards on final concentration of 10 μ g/mL resistance LB flat boards
Shaken in the identical resistance LB of 50mL training 12h, in the identical resistance LB of 200mL are seeded to by 2% inoculum concentration in shaken in 37 DEG C training fermentation
72h, takes the method for 30mL supernatant ammonium sulfate precipitations to extract crude protein, uses PBS soluble proteins, and dialysis removes salt ion.With sky
Carrier bacterial strain is control, and filter paper enzyme determines the fungistatic effect of the gene, finds 1366 bacterial strains to WB800, empty carrier bacterial strain, green grass or young crops
Rot opportunistic pathogen, the various bacteria such as bacterial leaf-blight opportunistic pathogen has certain fungistatic effect.Plasmid extraction is carried out to 1366 bacterial strains, and surveyed
Sequence is taken after gene order, is not compared in each large database concept and arrives any result, corresponding protein sequence is compared,
Do not compare result, it may be determined that the gene is new antifungal genes.
Listed above is only 2 specific embodiments of the present invention, and experiment material of the invention can be other plant, example
Such as garlic, corn edible crop, the medicinal plant such as honeysuckle, cordate houttuynia, can also be the biocontrol microorganisms such as Trichoderma harzianum.Antibacterial
The indicator bacteria selected in genescreen can also be fruit spot pathogen, ulcer pathogen, bacterial leaf streak of rice pathogen etc.
Various dead volume trophism pathogenetic bacterias.
Claims (2)
1. a kind of utilization bacillus subtilis expression system is quick, the method for high frequency zone antifungal genes, its step is：
1) preparation of the cDNA library containing target gene：
A. pathogen is inoculated with healthy plant, after the expression of induction of resistance gene, target plant total serum IgE is extracted；
B. mRNA is isolated and purified；
C. the Oligo dt of the restriction enzyme site containing Xba I are designed, its sequence is 5 '-ACAGGCTCTAGAGCTTTTTTTTTTTTTTTTT
TTTTTT-3 ', synthesizes first chain of cDNA, using first chain as templated synthesis Article 2 cDNA chains；After the completion of cDNA synthesis, plus
On the restriction enzyme site containing Nde I joint, described joint has 3 pairs, and sequence is as follows：
D.cDNA connection carriers：By the I pair of enzyme of restriction endonuclease Xba I and Nde of the cDNA for adding joint in step c
After cutting, it is connected on plasmid pBE-S；
E. linked system is transformed into efficient competent cell of escherichia coli, expands plasmid；Withered grass is converted after colony's upgrading grain
Bacillus WB800, no less than 2000 transformants of picking, is saved backup；
2) screening of lethal bacterial strain
A. transformant is shaken in the LB culture mediums of mark kalamycin resistance after training overnight, in point on the flat board of identical resistance
Sample, filters out script growing way normally but the later stage becomes the bacterial strain of transparent or semitransparent shape, preliminary to confirm, the gene has lethal work
With；
B. plasmid is extracted after the above-mentioned bacterium solution containing lethal gene tentatively confirmed being shaken into training, is converted again after being expanded through Escherichia coli withered
Careless bacillus WB800 and bacillus subtilis wild strain 168, occur in that lethal phenotype, show lethal phenotype by inserting
Gene cause, the gene as next step candidate gene；
3) screening of nematode resistance gene
Resistance of the transformant containing candidate gene to Caenorhabditis elegans is determined using filter paper enzyme, with bacillus subtilis
WB800 determines selection index as control, and selection exponential formula is as follows：Select index=(nematode population-control in test bacterium
Nematode population in bacterium)/line insect number, select transformant of the index less than -30% that there is anti-nematode to act on, the sieve for next step
Choosing；
4) screening of antifungal genes
Ammonium sulfate precipitation method extraction step 3) in the bacterial strain containing nematode resistance gene extracellular crude protein, using empty carrier bacterial strain as pair
According to filter paper enzyme determines the fungistatic effect of crude protein, extracting plasmid and gene sequencing, thus obtains corresponding antifungal genes.
2. utilization bacillus subtilis expression system according to claim 1 is quick, the method for high frequency zone antifungal genes,
It is characterized in that：Step 1) in linked system is transformed into efficient competent cell of escherichia coli, described competent cell
For E. coli competent HST08.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111171122A (en) * | 2020-01-07 | 2020-05-19 | 华中农业大学 | Antibacterial peptide PtR946 derived from pinellia ternata and application thereof |
| CN114426968A (en) * | 2022-02-22 | 2022-05-03 | 江南大学 | Bacillus subtilis full-length cDNA library construction method and directional screening application thereof |
| CN115028692A (en) * | 2022-06-22 | 2022-09-09 | 华中农业大学 | Bacillus subtilis antibacterial peptide BsR1 and application thereof |
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| US20060068397A1 (en) * | 2004-09-24 | 2006-03-30 | Xiongying Cheng | High throughput screening method for identifying molecules having biocidal function |
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| US20060068397A1 (en) * | 2004-09-24 | 2006-03-30 | Xiongying Cheng | High throughput screening method for identifying molecules having biocidal function |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111171122A (en) * | 2020-01-07 | 2020-05-19 | 华中农业大学 | Antibacterial peptide PtR946 derived from pinellia ternata and application thereof |
| CN111171122B (en) * | 2020-01-07 | 2021-06-01 | 华中农业大学 | Antibacterial peptide PtR946 derived from pinellia ternata and application thereof |
| CN114426968A (en) * | 2022-02-22 | 2022-05-03 | 江南大学 | Bacillus subtilis full-length cDNA library construction method and directional screening application thereof |
| CN114426968B (en) * | 2022-02-22 | 2024-03-26 | 江南大学 | Construction method of bacillus subtilis full-length cDNA library and directional screening application thereof |
| CN115028692A (en) * | 2022-06-22 | 2022-09-09 | 华中农业大学 | Bacillus subtilis antibacterial peptide BsR1 and application thereof |
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