CN108504672B - Ralstonia solanacearum N477 extracellular protein PHD and coding gene and application thereof - Google Patents
Ralstonia solanacearum N477 extracellular protein PHD and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses ralstonia solanacearum N477 extracellular protein PHD and a coding gene and application thereof. The amino acid sequence of the ralstonia solanacearum N477 extracellular protein PHD is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene thereof is shown as SEQ ID NO. 2. The PHD or the coding gene thereof is applied to the preparation of biological pesticide for resisting bacterial wilt. Designing a primer, using genome DNA as a template to amplify a PHD coding region sequence, connecting a product to a protein expression vector pET30a (+) vector, then transforming the product into escherichia coli BL21(DE3) for protein expression, and purifying PHD protein by using a nickel column. The greenhouse test result shows that the protein can also induce the tobacco to resist the infection of the tobacco mosaic virus, and the prevention effect is 56.88%.
Description
Technical Field
The invention belongs to the field of genetic engineering, and relates to ralstonia solanacearum N477 extracellular protein PHD (PopW Harpin domain), and a coding gene and application thereof.
Background
The hrp gene cluster of most gram-negative bacteria can cause anaphylactic reactions on non-host plants, while exhibiting pathogenicity on host plants. The product of the hrp gene plays a role in regulation and control in the expression and secretion process. Harpin proteins such as Erwinia amylovora HrpN, Pseudomonas syringae HrpZ and Ralstonia solanacearum PopA have been identified which are secreted extracellularly by a triple secretion system to cause allergic reactions in non-host plants, and are a type of protein rich in glycine, serine, lacking cysteine and resistant to heat, without similarity in amino acid sequence.
Bacterial wilt is a destructive soil-borne disease, is one of the plant diseases with the greatest harm, the widest distribution and the most serious loss in the world, and has no effective chemical pesticide and other prevention and control methods so far. Bacterial wilt is therefore called "cancer" of the plant. In recent years, the protected area in the north of China is obviously enlarged, and the production of vegetables in the plastic greenhouse in the south is enlarged, so that bacterial wilt in partial areas is generated and developed, even becomes a destructive disaster, and extremely serious economic loss is caused.
Ralstonia solanacearum can cause wilting of hundreds of crops all over the world and can infect more than 50 families and more than 200 plants, wherein the solanaceous plants are the most seriously damaged and comprise tomatoes, potatoes, tobaccos, peanuts, bananas and the like. The germs enter the xylem through the wound of the root of the host and then quickly spread to the whole plant. The pathogenic mechanism is that the ralstonia solanacearum can generate a large amount of extracellular polysaccharide when growing in the duct, can influence and hinder the water transportation in the plant body, and particularly easily blocks a stalk knot and a small-aperture duct perforated plate at a leaflet, thereby causing the withering of the plant. In model strain GMI1000, three extracellular proteins have been identified, PopA1, PopB and PopC, respectively. PopA1, a protein secreted extracellularly by the triple-type efflux system, lacks the N-terminal 92 amino acids. The derivative of PopA1, PopA3, has also been purified from the supernatant grown in nutrient-poor medium. Both PopA1 and PopA3 were able to elicit allergic reactions in petunia. The popA is located at 2kb upstream of the ralstonia solanacearum hrp gene cluster, and the expression of the popA is regulated and controlled by the hrpB gene. Arlat group found the novel proteins PopB and PopC from the genome of Ralstonia solanacearum, the PopB protein consisting of 173 amino acids and containing two nuclear localization signals; PopC is 1024 amino acids long, containing 22 leucine zippers. popB, popC and popA are all regulated by hrpB simultaneously. Under congo red induction, they found that PopB and PopC were secreted extracellularly by the Hrp system.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a ralstonia solanacearum N477 extracellular protein PHD.
Another objective of the invention is to provide a ralstonia solanacearum N477 extracellular protein PHD encoding gene.
The invention also aims to provide application of the ralstonia solanacearum N477 extracellular protein PHD coding gene.
The amino acid sequence of the ralstonia solanacearum N477 extracellular protein PHD is shown in SEQ ID No. 1.
The coding gene of the ralstonia solanacearum N477 extracellular protein PHD.
The preferable nucleotide sequence of the coding gene of the ralstonia solanacearum N477 extracellular protein PHD is shown as SEQ ID NO. 2.
And the recombinant expression vector contains the coding gene of the ralstonia solanacearum N477 extracellular protein PHD.
The gene engineering bacteria containing the coding gene of the ralstonia solanacearum N477 extracellular protein PHD.
The ralstonia solanacearum N477 extracellular protein PHD is applied to the preparation of a biological source pesticide for resisting bacterial wilt.
The coding gene of the ralstonia solanacearum N477 extracellular protein PHD is applied to the preparation of the biological pesticide for resisting the bacterial wilt.
The recombinant expression vector is applied to the preparation of biological pesticide for resisting bacterial wilt.
The genetic engineering bacteria are applied to the preparation of biological pesticide for resisting bacterial wilt.
The gene engineering application of the coding gene containing the ralstonia solanacearum N477 extracellular protein PHD comprises the following steps:
1) cloning of PHD Gene
Culturing ralstonia solanacearum N477 in a YGPA culture medium at the temperature of 30 ℃, and extracting ralstonia solanacearum genome DNA according to the operation instruction of the bacterial genome extraction kit when the strains grow to the logarithmic phase;
designing a primer for amplifying a coding gene of PHD, using genome DNA as a template, and using high-fidelity pfu polymerase to amplify the coding region sequence of the PHD gene, wherein the amplification procedure is as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, renaturation at 58 ℃ for 30s, extension at 72 ℃ for 1min, 30 cycles, and then cutting and recycling the product at 72 ℃ for 10 min; the primer for amplifying the PHD coding gene is selected from the group consisting of a primer 1: the upstream primer of the primer 1 is shown as SEQ ID NO.3, and the downstream primer of the primer 1 is shown as SEQ ID NO. 4;
2) expression and purification of PHD protein
Cutting the product, recovering the product, connecting the product to an intermediate carrier for enzyme digestion or connecting the product to a protein expression carrier pET30a (+) carrier after direct enzyme digestion, sequencing and identifying to obtain a recombinant carrier pET-PHD, then transforming the recombinant carrier into escherichia coli BL21(DE3) for protein expression, inoculating the screened positive clone into a test tube containing an LB liquid culture medium, culturing at 37 ℃ and 180rpm overnight, inoculating into 10ml of LB according to the proportion of 1:100 on the next day, culturing for 3h according to the above conditions, adding isopropyl-beta-D-thiogalactoside to make the final concentration 1mM, inducing and culturing for 2h, centrifuging at 4 ℃ and 6000rpm for 10min, collecting the thallus, suspending the thallus in 20mM Tris-HCl and pH 8.0, adding benzyl sulfonyl fluoride with the final concentration of 1mM, then crushing the thallus on an ultrasonic crusher, and when the bacterial liquid becomes clear, centrifuging to extract supernatant, performing polyacrylamide gel electrophoresis detection, and purifying PHD protein by using a nickel column by referring to the High-Affinity Ni-IDA Resin operation instruction to obtain purified PHD.
Advantageous effects
The PHD obtained by the inventor is a heat-resistant harpin protein and can cause tobacco anaphylactic reaction. PopW is an inactive pectinase homologue which can cause HR reaction, but the invention finds that PHD plays a decisive role, can more effectively inhibit TMV infection of tobacco and has obviously better effect than PopW.
3. The PHD treated tobacco obtained by the invention can induce the tobacco to resist the infection of tobacco mosaic virus, and the resistance is not limited to the PHD treated leaf blade, but also comprises the surrounding leaf blades, and the resistance is accompanied with the expression of PR-1 gene in the process. The PHD can induce the tobacco system to obtain disease resistance. This indicates that PHD has value for development into biogenic pesticides.
Drawings
FIG. 1 shows PHD protein expression results.
M is protein marker (SM0431, MBI), 1 is 1% BSA, 2 is the result of expression of the PopW clone, and 3 is the result of expression of the PHD clone.
Biological material preservation information
The N477 strain is classified and named as Ralstonia solanacearum of the genus Ralstonia of the family Solanaceae, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 2018, 1 month and 22 days, is preserved in the No.3 Hospital No.1 West Lu of Beijing city facing Yang area, is preserved in the microorganism research institute of China academy of sciences, and has the strain preservation number of CGMCC NO. 15253.
Detailed Description
Example 1
(1) Cloning of PopW Gene sequences
Ralstonia solanacearum N477 was cultured in YGPA medium (0.5% peptone, 0.5% yeast extract powder, 1% glucose and 1.5% agar) at 30 ℃ and Genomic DNA was extracted when the strain grew to logarithmic phase (Axyprep Bacterial Genomic DNA kit, Hangzhou). Designing an amplification primer according to a model bacterial ralstonia solanacearum GMI1000 whole genome sequence:
the upstream primer F1: ATGTCCATCCAGATTGATCGC (SEQ ID NO.4)
The downstream primer R1: GCCCGAGTAGGCCTTGTAG (SEQ ID NO.5)
Using N477 genome DNA as a template to carry out PCR amplification, wherein the PCR program is as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, renaturation at 60 ℃ for 1min, extension at 72 ℃ for 1min, 30 cycles, and electrophoresis at 72 ℃ for 10min, wherein the PCR amplification product of 1143bp is detected by 1% Agarose electrophoresis, and the product is recovered according to the Agarose Gel DNA Purification Kit (TaKaRa, Dalian) manual. And connecting 4 mu l of the recovered product to a vector pMD19-T, reacting for 16h at 16 ℃, transforming escherichia coli competence DH5 alpha by a heat shock method, picking positive clones for sequencing, and indicating that the complete coding region of the ralstonia solanacearum N477 extracellular protein gene PopW is 1143bp (SEQ ID NO.3) by a sequencing result.
(2) PopW protein expression
Designing primers with enzyme cutting sites at two ends according to the full-length coding sequence of the pseudomonas solanacearum N477 extracellular protein PopW gene:
the upstream primer F2: CG (CG)CATATGTCCATCCAGATTGATCGC Nde I(SEQ ID NO.6)
The downstream primer R2: GCAAGCTTGCCCGAGTAGGCCTTGTAG Hind III(SEQ ID NO.7)
And (3) performing PCR amplification on the coding region sequence of the popW gene by taking the genomic DNA of the N477 as a template, wherein the amplification program comprises the following steps: pre-denaturing at 94 ℃ for 4min, denaturing at 94 ℃ for 30s, renaturing at 60 ℃ for 1min, extending at 72 ℃ for 1min, after 30 cycles, at 72 ℃ for 10min, the size of the product is 1143bp, adding A to the tail end of the product recovered by cutting gel, connecting the product to a vector pMD19-T, cloning the product to a protein expression vector pET30a (+) by utilizing Nde I and Hind III enzyme cleavage sites, carrying out sequencing and identification to obtain a recombinant plasmid pET-PopW, then transforming the recombinant plasmid into BL21(DE3) for protein expression, centrifuging cultured escherichia coli BL21(DE3) containing the recombinant plasmid, collecting thalli, suspending the thalli in 20 mM-Tris (pH 8.0), adding a protease inhibitor PMSF with the final concentration of 1mM, carrying out protein disruption on an ultrasonic disruptor, then extracting the protein, carrying out polyacrylamide gel electrophoresis detection, and purifying the PopW protein by using a nickel column.
(3) Cloning of PHD
Designing an amplification primer according to the full-length coding sequence of the pseudomonas solanacearum N477 extracellular protein PopW gene:
the upstream primer F3: GGTCGGCTCGGGCGGCTT (SEQ ID NO.8)
The downstream primer R3: TCCATCCAGATTGATCGCCCG (SEQ ID NO.9)
PCR amplification was performed using N477 genomic DNA as a template, and the PCR procedure was as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, renaturation at 58 ℃ for 30s, extension at 72 ℃ for 1min, 30 cycles, and electrophoresis at 72 ℃ for 10min, wherein the PCR amplification product is 454bp detected by 1% Agarose electrophoresis, and recovered according to the Agarose Gel DNA Purification Kit (TaKaRa, Dalian) manual. Connecting 4 mu l of the recovered product to a vector pMD19-T, reacting for 16h at 16 ℃, transforming escherichia coli competent DH5 alpha by a heat shock method, selecting a positive clone for sequencing, and the sequencing result shows that the complete coding region of the N477 extracellular protein gene PHD is 441 bp.
(4) PHD protein expression
Designing primers with enzyme cutting sites at two ends according to the full-length coding sequence of the pseudomonas solanacearum N477 extracellular protein PopW gene:
the upstream primer F4: ATAAGCTTGGTCGGCTCGGGCGGCTT Hind III(SEQ ID NO.10)
The downstream primer R4: GCCATATGTCCATCCAGATTGATCGCCCG Nde I(SEQ ID NO.11)
Taking N477 genome DNA as a template, and amplifying the coding region sequence of the PHD gene by PCR, wherein the amplification procedure is as follows: pre-denaturation at 94 deg.C for 4min, denaturation at 94 deg.C for 30s, renaturation at 58 deg.C for 30s, extension at 72 deg.C for 1min, 30 cycles, cloning the product to an intermediate vector pMD19-T with the size of 454bp at 72 ℃ for 10min, further cloning to a protein expression vector pET30a (+) vector by utilizing the introduced Nde I and Hind III enzyme cutting sites, sequencing and identifying to obtain a recombinant plasmid pET-PHD, then transformed into BL21(DE3) for protein expression, cultured Escherichia coli BL21(DE3) containing the recombinant plasmid is centrifuged to collect the cells, the cells are suspended in 20mM Tris-HCl (pH 8.0), and a protease inhibitor PMSF is added to the cells at a final concentration of 1mM, protein disruption was performed on an ultrasonicator, followed by extraction of the protein, detection by polyacrylamide gel electrophoresis, and purification of PHD protein using a nickel column to obtain a protein concentration of 25mg/LW (FIG. 1).
Example 2
The PopW soluble protein and PHD protein obtained above were sprayed onto tobacco leaves by spraying, and sterilized water and empty vehicle containing no PHD protein were used as controls. After two days, the leaves sprayed with PHD and two leaves above and below the leaves were inoculated with tobacco mosaic virus by the friction inoculation method, 3 times for each treatment, and 12 tobacco seedlings for each treatment. After 4 days, the number of the disease spots is observed, and the control effect is calculated through the number of the disease spots. The control effect is equal to [ (the number of the scorched spots on the control leaves-the number of the scorched spots on the treated leaves)/the number of the scorched spots on the control leaves ] x 100%. The results show that compared with the blank control without treatment, the number of the disease spots on the leaves treated by the PopW soluble protein and the PHD is obviously reduced, and the control effect can respectively reach 45 percent and 56.88 percent (Table 1), which indicates that the PopW soluble protein and the PHD can cause systemic resistance of tobacco, but the PHD can more effectively inhibit the TMV infection of the tobacco, thereby effectively inhibiting the tobacco mosaic virus infection.
TABLE 1 biocontrol effects of PHD on tobacco mosaic Virus
Note: and recording the number of the dead spots on each diseased leaf after 4 days of treatment, and calculating the inhibition rate of the dead spots, namely the protein control effect.
The above examples show that the novel extracellular protein PHD of Ralstonia solanacearum N477 cloned in the invention has Harpin characteristics, including amino acid composition, thermal stability, protease sensitivity and slow migration on SDS-PAGE, and both PHD and PopW can cause HR reaction. The PHD treated tobacco of the invention can induce the tobacco to resist the infection of the tobacco mosaic virus, and the resistance is not limited to the PHD treated leaf and the surrounding leaf, and the expression of PR-1 gene is accompanied in the process. The PHD can induce the tobacco system to obtain disease resistance, and the PHD can be developed into biogenic pesticide.
Claims (3)
1. The application of the coding gene of ralstonia solanacearum N477 extracellular protein PHD in preparing a biological source pesticide for resisting tobacco mosaic virus; the coding gene nucleotide sequence of the ralstonia solanacearum N477 extracellular protein PHD is shown as SEQ ID No. 2.
2. The application of the recombinant expression vector containing the coding gene of the ralstonia solanacearum N477 extracellular protein PHD in preparing the tobacco mosaic virus disease resistant biological pesticide is disclosed, wherein the nucleotide sequence of the coding gene of the ralstonia solanacearum N477 extracellular protein PHD is shown as SEQ ID No. 2.
3. The application of the genetic engineering bacteria containing the coding gene of the ralstonia solanacearum N477 extracellular protein PHD in preparing the biological source pesticide for resisting the tobacco mosaic virus disease is disclosed, wherein the nucleotide sequence of the coding gene of the ralstonia solanacearum N477 extracellular protein PHD is shown as SEQ ID No. 2.
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