CN116334036B - Method for screening bacterial wilt-resistant extracellular nuclease from bacterial wilt and genetic improvement application - Google Patents
Method for screening bacterial wilt-resistant extracellular nuclease from bacterial wilt and genetic improvement application Download PDFInfo
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- CN116334036B CN116334036B CN202211004364.8A CN202211004364A CN116334036B CN 116334036 B CN116334036 B CN 116334036B CN 202211004364 A CN202211004364 A CN 202211004364A CN 116334036 B CN116334036 B CN 116334036B
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Abstract
The invention belongs to the technical field of genetic transformation, and particularly relates to a method for screening bacterial wilt-resistant extracellular nuclease from bacterial wilt and genetic improvement application. The result of gene knockout of the nuclease shows that the phenotype of the bacterial wilt is changed after the knockout, the pathogenicity is reduced, and the competitiveness with a wild strain is also reduced. In the embodiment, ruvC is over-expressed in the infected tomato variety MoneyMarker, the transformed plant can obviously improve the resistance of the tomato to bacterial wilt, is favorable for breeding of the tomato resistant variety, is also successfully expressed in arabidopsis and tobacco, does not influence the normal growth of crops, and has the potential of being applied to disease-resistant breeding of more crops.
Description
Technical Field
The invention belongs to the technical field of genetic transformation, and particularly relates to a method for screening bacterial wilt-resistant extracellular nuclease from bacterial wilt and genetic improvement application.
Background
Bacterial wilt of tomato is a bacterial soil-borne disease caused by Ralstonia solanacearum (Ralstonia solanacearum), one of the most serious bacterial diseases endangering agricultural production. Bacterial wilt invades from the root through plant wound or natural orifice, colonizes xylem, invades vascular bundles, hinders the transportation of moisture, and causes the plant to wilt and eventually die. The host of the bacterial wilt comprises hundreds of crops such as tomatoes, tobacco, potatoes and the like, the bacterial wilt has strong adaptability, and suitable selection pressure is provided for genetic variation of the bacterial wilt.
At present, the prevention and treatment research on tomato bacterial wilt mainly focuses on the aspects of disease-resistant variety breeding, agricultural prevention and treatment, biological prevention and treatment, chemical prevention and treatment and the like. The pesticide is mainly used for preventing and controlling in production, however, the drug resistance of the bacterial wilt is increased after long-term administration, the consumption is increased, the vicious circle is caused, and the long-term use of chemical pesticide is not friendly to the environment and the human health. The agricultural control can play a better role in preventing but can not effectively and fundamentally prevent the disease from expanding and spreading. In recent years, scientific researchers focus on screening and prevention effect evaluation of bacterial wilt biocontrol bacteria, however, from district tests to field tests can still be influenced by soil microorganisms, temperature and humidity, cultivation conditions and the like, so that the prevention effect difference of different areas is increased. In contrast, breeding disease-resistant varieties is an economic, safe and considerable strategy, and searching for disease-resistant related gene resources is one of important directions of bacterial wilt-resistant genetic breeding.
Disclosure of Invention
The invention aims to provide a method for screening bacterial wilt-resistant extracellular nuclease from bacterial wilt and genetic improvement application, and the screened extracellular nuclease RuvC is derived from the bacterial wilt and can be conveniently, safely and effectively applied to genetic breeding, so that the probability of resistance loss of resistant varieties is reduced to a certain extent.
The invention provides a method for screening bacterial wilt-resistant extracellular nuclease from bacterial wilt, which comprises the following steps: screening a binding protein related to an extracellular Holliday Junction structure from a bacterial wilt genome to obtain nuclease;
Knocking out the gene of the nuclease in the bacterial wilt genome, and when the phenotype of the gene knocked out bacteria is changed and the pathogenicity is reduced and the competitiveness with a wild strain is weakened, the nuclease is the extracellular nuclease RuvC for resisting the bacterial wilt.
Preferably, the bacterial wilt genome comprises a bacterial wilt model strain GMI1000 genome.
Preferably, the method of knockout comprises a natural transformation method.
Preferably, after the extracellular nuclease RuvC is obtained by screening, the method further comprises mixing the extracellular nuclease RuvC purified in vitro with a biological membrane, and verifying the inhibition of the biological membrane by the extracellular nuclease RuvC.
Preferably, the amino acid sequence of the extracellular nuclease RuvC is shown in SEQ ID NO. 1.
The invention also provides application of the encoding gene RuvC of the bacterial wilt-resistant extracellular nuclease obtained by screening by the method in construction of bacterial wilt-resistant species.
Preferably, the method of constructing an anti-bacterial wilt species comprises genetic transformation.
Preferably, the species of anti-bacterial wilt includes plants and microorganisms resistant to bacterial wilt.
Preferably, the plant is derived from the cruciferae and Solanaceae families.
The invention also provides a method for constructing the bacterial wilt-resistant species, which comprises the steps of transforming a coding gene RuvC, an expression cassette or an expression vector of the bacterial wilt-resistant extracellular nuclease into a target species, and constructing to obtain the bacterial wilt-resistant species;
The bacterial wilt-resistant extracellular nuclease is obtained by screening through the method.
The beneficial effects are that: according to the invention, nuclease aiming at extracellular Holliday Junction structure of bacterial wilt is screened from bacterial wilt genome according to the characteristic of extracellular deoxyribonucleic acid (eDNA); the result of gene knockout of the nuclease shows that the phenotype of the bacterial wilt is changed and the pathogenicity is reduced after the knockout, and the competitiveness with a wild strain is also reduced, which indicates that the screened nuclease RuvC for resisting the bacterial wilt can play a certain function in the pathogenic process of the bacterial wilt. In the embodiment, the nuclease activity of RuvC is also verified in vitro, and the RuvC is found to have degradation effect on biological membranes in vitro, so that the RuvC is over-expressed in a Money Marker of a disease-sensitive tomato variety, and the transformed plant has a certain bacterial wilt resistance. The result shows that the RuvC not only participates in the pathogenicity of the ralstonia solanacearum, but also has the application prospect of disease-resistant breeding. In the embodiment of the invention, ruvC is overexpressed in tomatoes, so that the resistance of tomatoes to bacterial wilt can be obviously improved, the breeding of tomato resistant varieties is facilitated, the expression is also successfully carried out in arabidopsis and tobacco, the normal growth of crops is not influenced, and the method has the potential of being applied to disease-resistant breeding of more crops.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a structural prediction result diagram (AlphaFold structure prediction) of RuvC;
FIG. 2 is a plasmid map of the pBlueScript SK+ vector;
FIG. 3 is a diagram of the result of RuvC knockout of the gene of interest, wherein A: PCR amplification result diagram; b: colony phenotype; c: a growth curve;
FIG. 4 is a graph of delta ruvc-related growth characteristics results, wherein A: a decrease in Δ ruvc extracellular polysaccharide levels; b: delta ruvc forms more biofilm;
FIG. 5 shows the results of the pathogenicity test of Delta ruvc on tomato susceptible varieties;
FIG. 6 is a graph showing the results of in vitro biofilm staining imaging and quantitative analysis of Delta ruvc;
FIG. 7 is a graph showing results of RuvC in vitro purification and biofilm treatment;
FIG. 8 is the effect of RuvC on plant resistance to bacterial wilt after overexpression of susceptible tomato Mao Genzhong;
FIG. 9 shows the traits of EPS depolymerization enzyme phiAP1_43 and RLS2_66 after MoneyMarker Mao Genzhong expression;
FIG. 10 is a exDNaseDor51_gp29 trait following Mao Genzhong expression;
FIG. 11 is a class HollidayJunction structurally related binding protein retrieved in the GMI1000 genome database;
FIG. 12 is a map of the plant over-expression vector pCAMBIA 2300;
FIG. 13 is a map of the bacterial wilt gene complementation vector pBBR 1-MCS-HA.
Detailed Description
The invention provides a method for screening bacterial wilt-resistant extracellular nuclease from bacterial wilt, which comprises the following steps: screening a binding protein related to an extracellular Holliday Junction structure from a bacterial wilt genome to obtain nuclease;
Knocking out the gene of the nuclease in the bacterial wilt genome, and when the phenotype of the gene knocked out bacteria is changed and the pathogenicity is reduced and the competitiveness with a wild strain is weakened, the nuclease is the extracellular nuclease RuvC for resisting the bacterial wilt.
The bacterial wilt genome of the present invention preferably comprises the bacterial wilt model strain GMI1000 genome. The invention utilizes the GMI1000 gene information all from the Ralstonia solanacearum sp (inra. Fr) database.
The binding proteins described herein that are structurally related to extracellular class Holliday Junction are all from the NCBI or Ralstonia solanacearum sp (inra. Fr) databases. Since the Holliday Junction-like binding proteins included two subfamilies of Integrated Host Factor (IHF) and histone-like protein (HU), the homologous genes of the two subfamilies were searched and aligned in the database described above to obtain six related binding proteins (FIG. 11).
After obtaining the nuclease according to the present invention, the nuclease gene is preferably knocked out, and the extracellular nuclease activity of the nuclease is verified, wherein the knockout preferably comprises a natural transformation method. The natural transformation method preferably comprises the steps of designing upstream and downstream fragments of primer clone RuvC, respectively having up and down lengths of about 600bp, carrying out electrophoresis and recovery after PCR amplification, and respectively connecting up and down to pBlueScript SK+ vectors with Spec resistance fragments to obtain pBlueScript SK-up-Spec-dn; transferring to MG1061 (JM 110), extracting plasmid DNA, performing double enzyme digestion to verify correctness, taking correct vector, extracting and purifying plasmid, and performing large-scale enzyme digestion and recovery on the extracted plasmid by using enzyme digestion site KpnI of RuvC-up-FP.
The plant over-expression vector skeleton used in the invention is pCAMBIA2300 (figure 12), and CaMV 35s promoter is connected by SalI/EcoRI double enzyme digestion on the basis of pCAMBIA2300 original plasmid in the early stage of a laboratory: MCS-HA-NOS terminator reading frame, pCAMBIA2300-35s: MCS-HA vector; cloning a target gene, firstly carrying out double-enzyme tangential linearization on a vector by utilizing BamHI/StuI on the basis of pCAMBIA2300-35s:MCS-HA, then predicting a signal peptide sequence of tomato SlPR1 through a website SignaIP 5.0.0 (SignalP-5.0-Services-DTU HEALTH TECH), connecting the signal peptide sequence and a full-length sequence of a candidate gene RuvC on the linearized pCAMBIA2300-35s:MCS-HA to form the pCAMBIA2300-35s:SlPR1-RuvC-HA of the over-expression vector, and transferring the constructed correct plasmid into agrobacterium for standby by utilizing an agrobacterium electric shock transformation method, wherein the pCAMBIA2300-35s:MCS-HA is transferred into agrobacterium rhizogenes as a control of tomato hairy root transformation.
The invention compares the phenotype, pathogenicity and competitiveness of the gene knockout bacterium with that of a wild strain, for example, the phenotype of the gene knockout bacterium is changed, the pathogenicity is reduced, the competitiveness of the gene knockout bacterium with the wild strain is reduced, and the knocked-out gene is proved to be extracellular nuclease RuvC for resisting bacterial wilt.
The present invention preferably further comprises mixing the extracellular nuclease RuvC purified in vitro with a biological membrane after determining the extracellular nuclease RuvC, and verifying the inhibition of the biological membrane by the extracellular nuclease RuvC. Because RuvC is an extracellular nuclease, the activity of RuvC can be verified by in vitro purification, and if RuvC has an inhibitory effect on a biological membrane in vitro, ruvC can be used for target degradation of the biological membrane, and is overexpressed in host plants such as tomatoes, and whether the expressed plants have bacterial wilt resistance can be verified.
The extracellular nuclease RuvC has degradation effect on biological membranes in vitro, the amino acid sequence of the extracellular nuclease RuvC is preferably shown as SEQ ID NO.1, the nucleotide sequence of the encoding gene of the extracellular nuclease RuvC is preferably shown as SEQ ID NO.2, and the extracellular nuclease RuvC is predicted to have a structure shown as shown in FIG. 1.
The invention also provides application of the encoding gene RuvC of the bacterial wilt-resistant extracellular nuclease obtained by screening by the method in construction of bacterial wilt-resistant species.
The method for constructing the bacterial wilt-resistant species preferably comprises genetic transformation, the specific method of the genetic transformation is not particularly limited, and the genetic transformation can be carried out by utilizing the conventional method in the field to obtain a transgenic line with stable expression.
The bacterial wilt resistant species preferably comprises bacterial wilt resistant plants and microorganisms, the plants are preferably derived from cruciferae and solanaceae, as in the embodiment of the invention, the RuvC gene shown in SEQ ID NO.2 is transformed into tomato, arabidopsis thaliana and tobacco by utilizing a genetic transformation mode, and the transformed plants have certain bacterial wilt resistant capability, do not influence the normal growth of crops, and have the potential of being applied to disease resistance breeding of more crops.
The invention also provides a method for constructing the bacterial wilt-resistant species, which comprises the steps of transforming a coding gene RuvC, an expression cassette or an expression vector of the bacterial wilt-resistant extracellular nuclease into a target species, and constructing to obtain the bacterial wilt-resistant species;
The bacterial wilt-resistant extracellular nuclease is obtained by screening through the method.
The method of the transformation is not particularly limited and may be any method conventionally used in the art. As in the present examples, agrobacterium rhizogenes mediated tomato hairy root transformation and agrobacterium mediated tomato genetic transformation are employed, and the agrobacterium rhizogenes mediated tomato hairy root transformation is specifically described, preferably comprising the steps of:
1) Selecting about 100 full and healthy Money Marker seeds, pouring the seeds into a 50mL centrifuge tube, washing the seeds for 5min by using 75% alcohol, pouring the alcohol, washing the seeds for 3 times by using sterilized water, and then using 50% sodium hypochlorite solution (84 disinfectant: distilled water = 1:1) for 5min, the process is performed in an ultra clean bench; pouring out the sodium hypochlorite solution, and cleaning the seeds with sterilized water for 3 times; the washed seeds were placed on the surface of 1/2MS medium and cultured in a tissue culture chamber for about 14d until the first true leaves began to grow.
2) Cutting off the root of the tomato, dipping a single colony of agrobacterium rhizogenes expressing a target gene (pCAMBIA 2300-35s: slPR1-RuvC-HA) by using a sterilizing gun head, placing the tomato back to a 1/2MS flat plate, and covering a layer of wet filter paper on the base part of the hypocotyl; after 1-2 weeks, the tomatoes develop new roots, the roots are cut off with a scalpel (the roots are false positive at this time), the tomatoes are returned to the 1/2MS plate and a layer of moist filter paper is placed on the base of the hypocotyl to continue rooting.
3) After cutting off the first growing roots, seedlings were placed on a slant solid medium containing antibiotics (a layer of filter paper was laid on the medium in advance), and a layer of moist filter paper was covered on the hypocotyl wound, and new roots were observed at 14-18 days, and agrobacterium containing pCAMBIA2300-35s: slpr1-RuvC-HA vector was selected as a control.
4) Selecting tomato roots on a slant culture medium for sampling, performing WesternBlot detection, transplanting positive seedlings into nutrient soil, and performing moisture preservation culture.
The agrobacterium-mediated tomato genetic transformation in the examples of the invention preferably comprises the following steps:
1) Sowing: selecting about 100 full and healthy Money Marker seeds, pouring the seeds into a 50mL centrifuge tube, washing the seeds with 75% alcohol for 5min, pouring the alcohol, washing the seeds with sterilized water for 3 times, and washing the seeds with 50% sodium hypochlorite solution (84 disinfectant: distilled water=1:1) for 5min, wherein the process is carried out in an ultra-clean workbench; pouring out the sodium hypochlorite solution, and cleaning the seeds with sterilized water for 3 times; the cleaned seeds are placed on the surface of a 1/2MS culture medium, and are cultured for about 10 days in a tissue culture chamber until two cotyledons are fully developed.
2) Preparation of explants: placing a piece of filter paper in a culture dish, wetting the filter paper with sterile ddH 2 O, taking out the sterile seedling with cotyledon spread, cutting the tip and petiole of the cotyledon, cutting into 1 piece of explant, placing on KCMS culture medium (a layer of filter paper is placed on the culture medium), and culturing in dark for 2d.
3) Preparing bacterial liquid: agrobacteria containing a target gene (2300-35 s: slPR1-RuvC-HA) are picked from the temperature of minus 80 ℃ and streaked on a Kan+Gen resistant LB plate for activation, inverted culture is carried out at the temperature of 28 ℃, single colony is picked at night in 2mL Kan+Gen resistant liquid LB medium at 28 ℃ and at 190r/min for shaking culture for 12-16 hours before the infection, and then 200 mu L of bacterial liquid is added into 10mL Kan+Gen resistant LB medium at 28 ℃ and at 190r/min for shaking culture for 6-8 hours.
4) Infection: pouring the activated bacterial liquid into a 15mL centrifuge tube, centrifuging at 25 ℃ for 20min at 4000r/min, pouring out the supernatant, and adding 4mL of agrobacterium suspension to suspend bacterial cells (the agrobacterium suspension must be opened in an ultra clean bench); measuring the OD 600 value of the bacterial liquid, and diluting the bacterial liquid to OD 600 =0.1-0.2; adding 30mL of suspension bacteria liquid into a culture dish, transferring all explants into the agrobacterium suspension liquid, and dip-dying for 3-4 min, and gently shaking the culture dish in the dip-dying process; pouring out the suspension, placing a piece of filter paper in a clean culture dish, transferring the explant onto the filter paper, and sucking the bacterial liquid as soon as possible; a layer of filter paper was laid on KCMS medium, and the explant was placed on the filter paper and co-cultured in dark environment for 2d.
5) Screening: transferring the explants to a 2Z culture medium, placing 90-100 pieces of explants on each culture dish, placing the leaves on the right side upwards, culturing in a tissue culture chamber, observing the explants at least twice a day, and changing the 2Z culture medium for 12-15 days after the explants grow up to ensure enough growth space, transferring part of the explants to a new 2Z culture medium, and removing the brown explants.
6) Subculture: transferring the explant (callus expands and adventitious bud grows) well grown on the 2Z culture medium to 0.2Z culture medium, and subculturing for about 15 d; when adventitious buds grow out of the leaf, the part of the leaf of the explant is cut off, only the callus and the adventitious buds are left, and the explant is placed in a new 0.2Z culture medium for continuous culture for about 15 d.
7) Rooting culture: when the adventitious bud grows to 2-3 cm, cutting off the adventitious bud from the root base, transferring the adventitious bud into a rooting culture medium, and inducing rooting.
8) Transplanting: when adventitious roots of seedlings grow well, taking out transformed seedlings, cleaning root culture medium, transplanting the transformed seedlings into nutrient soil (domestic soil: vermiculite=1:1), covering the nutrient soil for moisturizing for about 1 week, taking two small leaf discs for each plant after uncovering, quick-freezing and grinding by liquid nitrogen, adding 50 mu L of 2X SDS loading buffer (beta-mercaptoethanol and DTT are added), carrying out metal bath at 95 ℃ for 10min, centrifuging for 15min 12000r/min, taking 15 mu L of supernatant for carrying out WesternBlot detection protein expression, and culturing plants with target protein expression for propagation.
For further explanation of the present invention, a method for screening bacterial wilt-resistant extracellular nucleases from bacterial wilt and genetic improvement applications provided in the present invention will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
In the examples of the present invention, the test materials used, as described in no particular way, are commercially available products commonly used in the art.
1. Plant material
Tomato (Solanum lycopersicum) mainly used is the tomato susceptible variety Money Maker.
2. Vectors and strains
The plant expression vector pCAMBIA2300 (purchased from Abcam) used for construction of tomato transgenic plants; coli expression vector pGST-MCS (engineered from pGEX, purchased from Sigma-Aldrich) for protein purification; gene knockout vector pBlueScriptSK + (purchased at AgilentTechnologies); gene complementation vector pBBR1-MCS-HA (constructed by Kenneth Peterson laboratories);
BL21 (ESCHERICHIA COLI) for prokaryotic expression, MG1061 (ESCHERICHIA COLI) constructed by the vector, GV3101 (Agrobacterium tumefaciens) for tobacco and tomato gene expression are purchased from Wuhan Bai Weinuo & Lv Biotech Co., ltd, and MSU440 (Agrobacterium tumefaciens) for tomato root gene expression is from Shanghai plant stress biology research center Alberto Macho laboratory, china academy of sciences, GMI1000 (Ralstonia solanacearum) for pathogenic inoculation and gene knockout.
3. The main reagent comprises:
Reagents such as Taq, pfu DNA Polymerase, pfu Buffer, dNTPs and proteinase K are purchased from the whole gold biotechnology company; gold medal Mix TSE101 was purchased from the department of biotechnology, the restriction enzymes and T4 ligase were purchased from the New English (NEB) biotechnology company; the column type DNA recovery kit is purchased from Shanghai biological engineering company; inorganic salts such as isopropanol, absolute alcohol, chloroform and the like and organic solvents are purchased from national drug group limited company; peptone and casein hydrolysate in the medium were purchased from OXOID corporation. HA WESTERN Blot antibodies were purchased from Roche, cat. No. 12013819001 and formulated with 1 XPBST+5% Milk dilution 2000-fold.
4. The main culture medium comprises:
CPG medium: bactopeptone No.210g, acid hydrolyzed casein 1g, glucose 5g, dissolved in 1L ddH 2 O, and sterilized at 121℃for 20min. The solid medium requires an additional 15g of agar powder.
BG medium: tryptophan 10g, glucose 2.5g, acid hydrolyzed casein 1g, dissolved in 1L ddH 2 O, and sterilized at 121℃for 20min. The solid medium requires an additional 15g of agar powder.
MM medium: (NH 4)2SO4 0.5g,KH2PO4 3.4g,FeSO4 0.125.125 mg,1M KOH adjusted to pH=7.0, dissolved in 1L ddH 2 O, sterilized at 121℃for 20min, sterilized and then added with 20% glycerol and 5M MgSO 4. A solid medium requires an additional 15g of agar powder.
SMM semi-solid medium: tryptophan 0.1g, glucose 2.5g, EDTA-2Na 38mg, phosphate buffer 10mL, agar 3.5g, dissolved in 1L ddH 2 O, and sterilized at 121℃for 20min. Wherein phosphate buffer (200 mL): k 2HPO4 2.354g、KH2PO4, pH was adjusted to 7.0.
1/2MS medium: MS Salt 2.15g, MES 0.5g, dissolved in 800mL ddH 2 O, pH adjusted to 5.8 with 5M KOH, sucrose 5.0g, and fixed to 1L. The solid medium requires an additional 8g of agar powder.
KCMS medium (preculture, co-culture medium): macroelement (20X) 50mL, trace element (200X) 5mL, ferric salt (200X) 5mL, inositol (20 mg/mL) 5mL, vitamin B1 (1 mg/mL) 1.3mL,2,4-D (1 mg/mL) 200. Mu.L, KH 2 PO4 (100 mg/mL) 2mL, KT (1 mg/mL) 100. Mu.L, sucrose 30.0g, pH=5.8, ddH 2 O constant volume to 1L, agar 7.4g, and autoclaved at 121℃for 30min.
2Z medium (screening medium): macroelement (20X) 50mL, microelement (200X) 5mL, ferric salt (200X) 5mL, organic matter (200X) 5mL, sucrose 30.0g, pH=5.8, ddH 2 O constant volume to 1L, agar 7.4g,121 ℃ autoclaving 30min, IAA (1 mg/mL) 100 μL, zeatin (1 mg/mL) 2mL, kanamycin (100 mg/mL) 1mL, cephalosporin (300 mg/mL) 1.2mL are added when the temperature is cooled to 60 ℃.
0.2Z medium (subculture medium): macroelement (20X) 50mL, trace element (200X) 5mL, ferric salt (200X) 5mL, organic (200X) 5mL, sucrose 30.0g, pH=5.8, ddH 2 O to volume to 1L, agar 7.4g,121℃autoclaving for 30min, cooling to 60℃200. Mu.L of zeatin (1 mg/mL), kanamycin (100 mg/mL) 1mL, cephalosporin (300 mg/mL) 1.2mL are added.
Rooting medium: macroelement (20X) 50mL, trace element (200X) 5mL, ferric salt (200X) 5mL, organic (200X) 5mL, IBA (1 mg/mL) 2mL, sucrose 30.0g, agar 7.4g, adjust to pH=5.8, ddH 2 O constant volume to 1L, autoclave at 121℃for 30min, add kanamycin (100 mg/mL) 500 μl and cephalosporin (300 mg/mL) 1.2mL when cooling to 60 ℃.
5. The main instrument is as follows:
Electric shock conversion instrument (Eppendorf Eporator, eppendorf, germany), PCR instrument (Mastercyclernexus, eppendorf, germany), centrifuge (5810R, eppendorf, germany), shake incubator (MQL-61R, min spring), plant growth chamber (HP 1500GS-B, ruihua), electrophoresis instrument (EPS 600, shanghai energy), balance (JY 5002, shanghai constant level), ultra clean bench (DL-CJ-2 NDI, beijing east Lian Haer), water bath (XMTD 6000, beijing long wind), gel imager (Chemidoc XRS+, american BIO-RAD), single-lens (B700, nikon), ultra-micro-spectrometer (DS-11, beijing times bright), enzyme-labeled instrument (SPARK, switzerland), ultraviolet visible spectrophotometer (UV 1300, shanghai analysis instrument), laser confocal microscope (N-STORM, japanese kon).
EXAMPLE 1 knockout of the desired Gene RuvC
The up and down fragments of RuvC (RSc 0503) are designed, and the up and down fragment lengths are about 600 bp. The upstream and downstream primers RuvC-up-FP-KpnI(SEQ ID NO.3,5′-CGGGGTACCAGCAGAACGTGCGCCTG-3′)、RuvC-up-RP-EcoRI(SEQ ID NO.4,5′-CCGGAATTCCAGGCGACAGTCTACGCCT-3′)、RuvC-down-FP-BamHI(SEQ ID NO.5,5′-CGCGGATCCGAGCGCTCCCCAAACG-3′)、RuvC-down-RP-XbaI(SEQ ID NO.6,5′-TGCTCTAGAGCGCGATGATCTCGGAC-3′) of the up and down fragments were designed for corresponding PCR amplification. PCR reaction system: GMI1000 gDNA 1. Mu.L, gold medal Mix 45. Mu.L, primers 2. Mu.L each. PCR reaction procedure: pre-denaturation at 98℃for 3min; denaturation at 98℃for 30s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 45s; a total of 35 cycles; last extension at 72 ℃ for 10min; preserving at 12 ℃ for 10min.
The up and down fragments are respectively connected to pBluescript SK+ vector (figure 2), the up and down fragments are respectively connected to two sides of spec resistance fragment by adopting the thought of restriction enzyme molecular cloning, and are transformed to MG1061 (JM 110), plasmid DNA is extracted in a micro-quantity, after enzyme digestion sequencing verifies that the plasmid DNA is correct, the correct vector is taken for extracting and purifying the plasmid, and the extracted plasmid is subjected to massive enzyme digestion and recovery of linearization knockout vector by using the enzyme digestion site KpnI of up-FP.
Activating the wild strain GMI1000 on a BG plate, picking a single colony, shaking and culturing in a liquid MM (Minimal Medium) culture medium at 28 ℃, adding the recovered product of the linearization knockout carrier after culturing, mixing, coating the mixed bacterial liquid on a microporous filter membrane with the thickness of 0.45 mu m, and culturing at 28 ℃ for 48 hours. After 48 hours, bacterial pus grows on the filter membrane, bacterial pus is flushed by sterile water and coated on a spec-resistant BG plate, and after single bacterial colony grows, the bacterial colony is subcultured in a spec-containing liquid BG culture medium for three generations, and bacterial liquid PCR verification is carried out. PCR identification included the following: full length fragment of protogene, spec resistant fragment, and ralstonia solanacearum flagellin Rs-flic.
Full length sequence of RuvC was from Ralstonia solanacearum sp (inra. Fr) database, and full length cloning primer RsRuvC-FLFP-SpeI(SEQ ID NO.7,5′-CGGACTAGTATGCGCATCCTCGGCATCG-3′)、RsRuvC-FLRP-StuI(SEQ ID NO.8,5′-AAAAGGCCTGCCGACCAGCCGGCCG-3′) of RuvC gene was designed using PRIMER PREMIER software for PCR reaction. PCR reaction system: ralstonia solanacearum DNA. Mu.L, 10xTaq Buffer 1. Mu.L, 25mM MgCl 2 1. Mu.L, 10mM dNTP 0.1. Mu.L, and primers 0.1. Mu.L each, taq 0.1. Mu.L. PCR reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 60 ℃ for 30s; extending at 72 ℃ for 30s; a total of 35 cycles; last extension at 72 ℃ for 10min; preserving at 12 ℃ for 10min.
To verify whether the Spec resistance fragment was replaced at the correct position on the bacterial wilt genome, the primers Spec-FP (SEQ ID NO.9, 5'-ATGCGAACCACTTCATCC-3'), spec-RP (SEQ ID NO.10, 5'-GATGTTGCGATACTTCGC-3') for the Spec resistance fragment were designed using PRIMER PREMIER software. The RuvC-up+spec fragment was de-amplified using RuvC-up-FP-KpnI and Spec-RP, PCR reaction System: ralstonia solanacearum DNA. Mu.L, 10 xTaq Buffer 1. Mu.L, 25mM MgCl 2 1. Mu.L, 10mM dNTP 0.1. Mu.L, and primers 0.1. Mu.L each, taq 0.1. Mu.L. PCR reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 56 ℃ for 30s; extending at 72 ℃ for 2min; a total of 35 cycles; last extension at 72 ℃ for 10min; preserving at 12 ℃ for 10min.
Finally, in order to avoid the pollution of the knocked-out transformant, a bacterial wilt specific primer Rs-flic is utilized to verify whether the transformant is bacterial wilt. The full length sequence of Rs-flic was from the Ralstonia solanacearum sp (inra. Fr) database and PCR reactions were performed using PRIMER PREMIER software to design the proof primer Rsol-fliC-FP(SEQ ID NO.11,5′-GAACGCCAACGGTGCGAACT-3′)、Rsol-fliC-RP(SEQ ID NO.12,5′-GGCGGCCTTCAGGGAGGTC-3′) for Rs-flic. PCR reaction system: ralstonia solanacearum DNA. Mu.L, 10 xTaq Buffer 1. Mu.L, 25mM MgCl 2 1. Mu.L, 10mM dNTP 0.1. Mu.L, and primers 0.1. Mu.L each, taq 0.1. Mu.L. PCR reaction procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s; annealing at 56 ℃ for 30s; extending at 72 ℃ for 30s; a total of 35 cycles; last extension at 72 ℃ for 10min; preserving at 12 ℃ for 10min.
As shown in FIG. 3, delta ruvc #7, #8 failed to amplify the same band as GMI 1000; successful amplification of the up+spec resistant fragment and flagellin Rs-flic indicated that the original gene fragments of #7, #8 were successfully replaced by spec fragments and that there was no strain contamination problem (FIG. 3A).
Wild-type strains GMI1000 and Delta ruvc #7, #8 were diluted with sterile water to OD 600＝10-6 lg (1/trans), spread evenly on CPG+TTC+spec plates, incubated at 28℃for 3-5 d, and the bacterial plate phenotype was observed, colonies of Delta ruvc #7, #8 were significantly smaller and less mobile than GMI1000 (FIG. 3B). At the same time, GMI1000 and Delta ruvc #7 and #8 bacterial solutions with original OD 600 of 0.05 were cultured in CPG liquid medium respectively, at least three technical replicates were set for each strain, OD 600 was measured every 5-6 h and growth curves were drawn, and as a result, it was confirmed that the growth rate of Delta ruvc was not affected (C in FIG. 3).
Example 2: delta ruvc related growth characterization study
Extracellular polysaccharide level detection: picking single colonies of wild strains GMI1000 and Delta ruvc #7 and Delta ruvc #8, and culturing the single colonies for 14 to 16 hours in a 2mLCPG liquid culture medium at 28 ℃ and 190r/min overnight; adjusting the OD 600 of the bacterial liquid to 1, adding 50 mu L of the bacterial liquid into CPG liquid culture medium to a final volume of 5mL, and culturing at 28 ℃ for 48h at 190 r/min; centrifuging at room temperature for 15min at 5000r/min, and removing thalli; mixing 100 μl of supernatant with 200 μl of acetylacetone-Na 2CO3 solution, adding 200 μl ddH 2 O, metal-bathing at 95deg.C for 10min, and cooling; adding 400 mu L of absolute ethyl alcohol and 200 mu L of p-dimethylaminobenzaldehyde solution, finally adding 900 mu L of absolute ethyl alcohol, shaking and mixing uniformly, standing at room temperature for 20min, and measuring the absorbance of the solution at 525 nm. As a result, as shown in fig. 4a, both Δ ruvc #7, #8 significantly reduced the level of EPS produced.
Biofilm formation ability assay: the cultured bacterial cells were collected by centrifugation, resuspended in fresh CPG medium, OD 600 was adjusted to 0.1, and added to a cell culture plate sealed with a gas permeable membrane, and incubated at 28℃for 3d. The culture broth was aspirated along the well walls, the well plates were washed 2 times with 100. Mu.L of sterile ddH 2 O and dried. Each well was fixed with 100. Mu.L of methanol for 10min, and then excess methanol was aspirated and air-dried at room temperature. 200. Mu.L of 0.1% (W/V) crystal violet was used to stain for 15-20 min at room temperature, excess crystal violet was aspirated, and the well plate was rinsed with 200. Mu.L of sterile water. The attached crystal violet was dissolved in 200. Mu.L of 95% ethanol at 37℃for 30 minutes, and the absorbance at 590nm was measured using an enzyme-labeled instrument.
The assay results are shown in FIG. 4B, where the strain formed more biofilm in vitro after the RuvC knockdown.
Example 3: detection of pathogenic ability of Delta ruvc on tomato susceptible varieties
The Delta RuvC knockout mutant obtained in example 1 was subjected to gene complementation of Delta RuvC, and the RuvC self Promoter and full length sequence were cloned using pBBR1-MCS-HA vector (FIG. 13) as a backbone, and the RuvC Promoter and full length sequence were obtained from Ralstonia solanacearum sp. (inra. Fr) database, and a primer RsRuvCpro_FP：(SEQ ID NO.13,5′-TAAAGGGAACAAAAGCTGAGGCGCAGAACGCGGGC-3′)、RsRuvC-HA-RP(SEQ ID NO.14,5′-TGGAACGTCGTATGGGTAGCCGACCAGCCGGCCG-3′),PCR reaction system of RuvC promoter+gene was designed using PRIMER PREMIER software: GMI1000 gDNA 1. Mu.L, gold medal Mix 45. Mu.L, primers 2. Mu.L each. PCR reaction procedure: pre-denaturation at 98℃for 3min; denaturation at 98℃for 30s; annealing at 55 ℃ for 30s; extending at 72 ℃ for 1min; a total of 35 cycles; last extension at 72 ℃ for 10min; preserving at 12 ℃ for 10min. The fragment obtained by PCR reaction is recombined on a pBBR1-MCS-HA carrier by utilizing a homologous recombination mode, and the homologous recombination system is as follows: pBBR1-MCS-HA vector 1. Mu.L, ruvC promoter+gene fragment 3. Mu.L, 5x CE II Buffer 2. Mu.L, exnase II. Mu. L, ddH 2 O3. Mu.L. The constructed pBBR1-pro is transformed into Delta RuvC-7 bacterial wilt competent cells by electric shock, the cells after electric shock transformation are coated on a flat plate of BG+antibiotics+TTC for screening, mature bacterial wilt single colonies are grown after 3-5 days, after single colony is picked for activation, western Blot is used for detection, the protein expression of the RuvC in the anaplerotic strain is successfully detected by utilizing an HA antibody (A in figure 5), which shows that the RuvC is successfully complemented.
Naturally root irrigation and inoculation: picking single colonies of the wild strains GMI1000 and Delta ruvc #7 and Delta ruvc #8, and shake culturing for 14-16 h at 28 ℃ and 190r/min in 2mL CPG liquid culture medium; adding 300 mu L of bacterial liquid into a 30mLCPG liquid culture medium, and performing expansion culture at the temperature of 28 ℃ and the speed of 190r/min for 10-12 hours; collecting bacterial liquid into a 50mL centrifuge tube, centrifuging at 4000r/min to remove supernatant, adding equal volume of sterile water to resuspend bacterial cells, measuring OD 600 value, and adding water to dilute until OD 600 value is 0.1; and (3) selecting plants growing for 4 weeks for inoculation, and filling 15ml of bacterial wilt bacterial liquid into the soil along the root of the soil surface, wherein each bacterial strain is inoculated for 6-8 times. The inoculated plants were placed in a light incubator at 28℃and 99% RH, 12h light/12 h dark for moist culture, and the disease condition of the plants was observed daily (B in FIG. 5).
According to the leaf wilting degree of the plants, the plant leaves are divided into 5 disease grades: grade 0, no wilting symptoms; stage 1, leaf wilting of 1% -25%; stage 2, leaf wilting of 26-50%; grade 3, 51% -75% leaf wilting; grade 4, 76% -100% leaf wilting, calculating and recording morbidity and disease index (C and D in figure 5), taking stem samples 5D after inoculation for counting bacterial growth, and the results show that delta ruvc-7 after inoculation has bacterial growth significantly lower than wild type GMI1000, while the bacterial growth in the plant inoculated with the anaplerotic strain has no significant difference from GMI1000 (E in figure 5).
Wherein N0, N1, N2, N3 and N4 represent the plant numbers with the disease grades of 0,1,2,3 and 4 respectively.
Knock-out mutant adaptive vaccination: equal amounts of Delta ruvc #7, #8 bacterial liquid and wild type bacterial strain GMI1000 bacterial liquid are mixed, and diluted to OD 600 of 0.01; selecting plants growing for 4 weeks, cutting off the 1 st true leaf, inoculating the petioles along the wound, and inoculating 2 mu L of mixed bacterial liquid to the petioles of each plant; after 3d, stem bacterial wilts were isolated, diluted and spread on CPG plates, and the number of each colony and the percentage of each strain to the total bacterial growth were counted, and bacterial growth of Delta ruvc #7, #8 in the plants was significantly lower than GMI1000, meaning that Delta ruvc was less adaptable and the competitiveness was decreased (F in FIG. 5).
Example 4: delta ruvc in vitro biofilm staining imaging and quantitative analysis
Wild strains GMI1000 and Delta ruvc #7, #8 biofilms were cultured in cell culture slides, the culture medium was changed every 24 hours at 28℃for 3 days, the excess medium was removed after the biofilms were mature, stained with the nucleic acid fluorescent dye LIVE/DEAD Baclight TM Bacterial Viability Kit, and observed under a laser confocal microscope after sealing. And analyzing the obtained images by COMSTAT software to obtain indexes such as biological membrane biomass, coverage rate, average thickness, average diffusion distance and the like, and comparing the phenotype of the wild strain GMI1000 biological membrane with the phenotype and the differences of various indexes of the delta ruvc #7 and #8 biological membranes under a confocal microscope.
As a result, as shown in fig. 6, Δ ruvc #7 and #8 formed thicker biofilms (a in fig. 6), and it was found by quantitative analysis that although Δ ruvc was less different in biomass than GMI1000 (B in fig. 6), both Δ ruvc #7 and #8 covered areas (C in fig. 6) and the average thickness distribution (D in fig. 6) were significantly higher than GMI1000.
Example 5: ruvC in vitro purification and biofilm treatment
The full-length gene of RuvC is connected with a GST label carrier, and after enzyme digestion sequencing verification is successful, the plasmid is transferred into the escherichia coli BL21, and a small amount of induced expression is performed first, so that the escherichia coli strain with the successfully induced expression is left. Amplifying and culturing a small amount of successfully induced bacterial liquid in an LB liquid culture medium, culturing at 37 ℃ until OD 600 = 0.92, adding IPTG with the final concentration of 0.25mM, and inducing at low temperature; after the induction, the bacterial liquid is crushed by ultrasonic until the bacterial liquid becomes clear, the supernatant is centrifugally reserved after the bacterial liquid is crushed, the supernatant is adsorbed at a low temperature of GSTbeads, and is eluted by 1X GST Solution Buffer after the adsorption is finished, the concentration of the first elution is 14.2mg/mL, and the concentration of the second elution is 3.3mg/mL (A in figure 7).
The eluted proteins were treated with final concentrations of 0.25mg/ml, 0.5mg/ml, 1mg/ml, and 2mg/ml, respectively, to treat bacterial wilt biofilms cultured in cell well plates, and after incubation at 37 ℃, excess culture material was removed and the biofilm content was measured by crystal violet method (FIG. 7B), and was significantly reduced when 0.5mg/ml of RuvC protein was added (FIG. 7C).
Example 6: influence of RuvC on plant resistance to bacterial wilt after overexpression of susceptible tomato Mao Genzhong
Sowing a tomato infected variety Money Marker on a 1/2MS sterile plate, vertically culturing for 12-14D until true leaves begin to emerge, transversely cutting tomato seedlings along a lower stem, dipping single bacterial colony of agrobacterium rhizogenes (MSU 440) capable of successfully expressing RuvC genes by using a transverse plane, placing the single bacterial colony on the 1/2MS plate after dipping, growing new roots after 6-7D, namely false positive, cutting the first-generation hairy roots, transferring the first-generation hairy roots into a resistant 1/2MS plate for rooting continuously, transferring the second-generation hairy roots into Jiffypot nutrient soil after growing, taking a proper amount of roots for WesternBlot detection protein expression (A in fig. 8), taking positive plants with correct expression for culturing, inoculating OD 600 after growing four true leaves of tomatoes, diluting to 0.01 GMI1000 fresh bacterial liquid, carrying out daily statistical observation on the disease conditions (B in fig. 8) and the index (C in fig. 8), taking a sample of the first-generation hairy roots after 5D after inoculating, diluting and coating the sample on the CPG plate for carrying out bacterial growth, and carrying out statistical counting after 3D in the CPG plate, and carrying out bacterial growth (GME in fig. 8), and obviously reducing the disease conditions after the bacterial growth index (GME in fig. 8) is remarkably and remarkably increasing the bacterial growth rate.
Comparative example 1
By the same method as in example 6, several EPS depolymerases and exDNases were overexpressed in the Money Marker Mao Genzhong of the infected tomato variety, and the plants with correct protein expression were transplanted and cultured, inoculated with GMI1000, and observed for disease.
The results showed that there was no significant difference in the incidence of EPS depolymerase phiAP1_43, rls2_66 after expression in Money Marker Mao Genzhong (fig. 9 a), compared to GMI1000 (fig. 9B), no decrease in the disease index of the transformed tomatoes, and no significant difference in the bacterial growth of tomato stems after 5D of inoculation (fig. 9D), even after exceeding GMI1000 in the post-inoculation period (fig. 9C).
ExDNaseDor. Mu.51 gp29 showed no apparent bacterial wilt resistance phenotype after Mao Genzhong expression, exDNase RusA had a reduced tendency to develop disease (B in FIG. 10), oxRusA had a slightly lower disease index than GMI1000 by statistical average disease index, oxDor. Mu.51 gp29 had no significant difference from GMI1000 (C in FIG. 10), and bacterial growth in tomato stems was found to be not reduced by 5D after inoculation, either Dor51. Mu.29 or RusA, compared to GMI1000 (D in FIG. 10). It was demonstrated that exDNase Dor51_gp29 had no anti-bacterial wilt effect after expression of tomato Mao Genzhong, and RusA had a weak tendency to anti-bacterial wilt, but was not as remarkable as RuvC.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (7)
1. A method for screening bacterial wilt resistant extracellular nucleases from bacterial wilt comprising the steps of: screening a binding protein related to an extracellular Holliday Junction structure from a bacterial wilt genome to obtain nuclease;
Knocking out the gene of the nuclease in the bacterial wilt-resistant genome, and when the phenotype of the gene knocked-out bacteria is changed and the pathogenicity is reduced and the competitiveness with a wild strain is weakened, the nuclease is the bacterial wilt-resistant extracellular nuclease RuvC, and the amino acid sequence of the extracellular nuclease RuvC is shown as SEQ ID NO. 1.
2. The method of claim 1, wherein the bacterial wilt genome comprises a bacterial wilt-model strain GMI1000 genome.
3. The method of claim 1, wherein the method of knockout comprises a natural transformation method.
4. The method of claim 1, further comprising mixing the in vitro purified extracellular nuclease RuvC with a biological membrane after screening for the extracellular nuclease RuvC, and verifying inhibition of the biological membrane by the extracellular nuclease RuvC.
5. Use of RuvC, which is a coding gene of bacterial wilt-resistant extracellular nuclease obtained by screening according to any one of claims 1 to 4, in the construction of bacterial wilt-resistant tomatoes.
6. The use according to claim 5, wherein the method of constructing a bacterial wilt-resistant tomato comprises genetic transformation.
7. The method for constructing the bacterial wilt-resistant tomato is characterized by comprising the steps of transforming a coding gene RuvC, an expression cassette or an expression vector of an extracellular nuclease for resisting bacterial wilt into a target species, and constructing to obtain the bacterial wilt-resistant tomato;
the bacterial wilt-resistant extracellular nuclease is obtained by screening according to the method of any one of claims 1 to 4.
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