CN104311646A - Application of recombinant protein HarpinZDelta254-298 - Google Patents

Application of recombinant protein HarpinZDelta254-298 Download PDF

Info

Publication number
CN104311646A
CN104311646A CN201410510546.1A CN201410510546A CN104311646A CN 104311646 A CN104311646 A CN 104311646A CN 201410510546 A CN201410510546 A CN 201410510546A CN 104311646 A CN104311646 A CN 104311646A
Authority
CN
China
Prior art keywords
hrpz
harpinz
protein
recombinant protein
psgs1
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201410510546.1A
Other languages
Chinese (zh)
Inventor
高学文
张阳
朱青青
伍辉军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Agricultural University
Original Assignee
Nanjing Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Agricultural University filed Critical Nanjing Agricultural University
Priority to CN201410510546.1A priority Critical patent/CN104311646A/en
Publication of CN104311646A publication Critical patent/CN104311646A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dentistry (AREA)
  • Environmental Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Pest Control & Pesticides (AREA)
  • Agronomy & Crop Science (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Gastroenterology & Hepatology (AREA)

Abstract

The invention discloses application of recombinant protein HarpinZDelta254-298. Functional segments HarpinZDelta254-298 of a HarpinZPsgS1 protein are obtained; the protein can be stably expressed in Escherichia coli in vivo, and has better biological effects on plants than full-length HarpinZPsgS1. After the two proteins with the same concentration are used for treating tobacco and rice, the HarpinZDelta254-298 have obviously higher effects of resisting mosaic virus for tobacco and promoting growth for rice than the full-length HarpinZPsgS1, and thus, can be used for enhancing the effects of resisting tobacco mosaic virus for tobacco and promoting growth for rice.

Description

Recombinant protein HarpinZ Δ 254-298application
Technical field
The invention belongs to genetically engineered field, relate to recombinant protein HarpinZ Δ 254-298application.
Background technology
Harpin albumen is the class protein exciton produced by plant pathogenetic bacteria, non-host plant can be excited to produce hypersensitive cell death ((hypersensitive cell death, HCD), inducing plant produces defense response, Promoting plant growth.These useful effects of Harpin albumen, may be realized by the different functional domain of protein, therefore be separated, identify in Harpin albumen most important with disease-resistant, that growth-promoting is relevant functional area.
In pseudomonas syringae belongs to, research for Kidney bean pvs oryzae and oryzicola P.syringae pv.phaseolicola functional domain is the most thorough, think that C-end exercises most important (M.Haapalainen et al to its function, 2011), but wherein physico-chemical property research is only confined to the research of functional domain, as polypeptide binding site, the aspects such as the formation of plasmalemma contaminated with lipid position and hole, lack the research to the disease-resistant aspect of plant growth-promoting.Therefore, functional area relevant to the useful effect on plant such as plant disease-resistant, growth-promoting in research Harpin albumen is significant, for the application of harpin albumen in agriculture production provides basis.This laboratory is cloned into the hrpZ gene that size is 1038bp early stage from cloves false unit cell soybean pvs oryzae and oryzicola (Pseudomonas syringae pv.glycinea) S1 bacterial strain, and utilizes escherichia coli expression.Research shows, Harpin psgS1there is the effect of evoking tobacco disease resistance and growth-promoting.The present invention on this basis, by research Harpin psgS1the functional domain of the biological effects such as middle decision HR reaction, disease-resistant, growth-promoting, obtains short peptide stretch that is disease-resistant, growth-promoting better effects if, is easier to vivoexpression, improves output and cost-saving, is beneficial to the industrialization of microbial proteinous agricultural chemicals.
Summary of the invention
The object of this invention is to provide a kind of recombinant protein HarpinZ Δ 254-298application.
Object of the present invention realizes by following technical scheme:
The recombinant protein HarpinZ of aminoacid sequence as shown in SEQ ID NO.6 Δ 254-298improving the application in Resistance In Tobacco tobacco mosaic virus disease effect, promotion paddy growth.
Wherein, described recombinant protein HarpinZ Δ 254-298prepared by following steps:
(1) with DH5 α/pMD18-T-hrpZ psgS1the plasmid DNA that bacterial strain extracts is template, uses upstream primer hrpZ Δ 254-298-P1, downstream primer hrpZ Δ 254-298-P2 carries out Inverse PCR amplification, reclaim test kit with gel and the fragment of Inverse PCR amplification is reclaimed purifying, utilize T4DNA ligase enzyme that the fragment after reclaiming is carried out DNA molecular voluntarily after cyclisation, proceed to bacillus coli DH 5 alpha, to cut through enzyme and sequence verification obtains positive recombinant bacterial strain DH5 α/pMD18T-hrpZ Δ 254-298, wherein upstream primer hrpZ Δ 254-298-P1 sequence as shown in SEQ ID NO.3, downstream primer hrpZ Δ 254-298-P2 sequence is as shown in SEQ ID NO.4;
(2) positive recombinant bacterial strain DH5 α/pMD18-T-hrpZ Δ 254-298put forward plasmid DNA, be connected to after BamH1 and EcoR1 double digestion on expression vector pET30a (+), cut through plasmid PCR and enzyme after proceeding to e. coli bl21 and identify positive strain BL21/pET30a (+)-hrpZ Δ 254-298;
(3) LB culture medium culturing positive strain BL21/pET30a (+)-hrpZ is utilized Δ 254-298, and utilizing IPTG to induce expression of recombinant proteins, thalline obtains thick leach protein through ultrasonic disruption, obtains the recombinant protein HarpinZ of purifying after purifying Δ 254-298.
Beneficial effect:
1. present invention obtains a HarpinZ psgS1the function fragment HarpinZ of albumen Δ 254-298, be HarpinZ psgS1clip a recombinant protein after 254-298 amino acids, this albumen can in intestinal bacteria body stably express, after purifying, often liter of bacterium liquid can obtain pure protein and be about 50mg.
2. the recombinant protein HarpinZ that obtains of the present invention Δ 254-298plant has than total length HarpinZ psgS1better biological effect, after two albumen process tobaccos of concentration, paddy rice, HarpinZ Δ 254-298all significantly total length HarpinZ is better than to Resistance In Tobacco mosaic virus effect, paddy rice growth promoting effect psgS1.
3. the truncated segment HarpinZ that obtains of the present invention Δ 254-298biological effect useful on plant confirms that the different zones of Harpin albumen regulates and controls different functions, important reference value is had for announcement Harpin protein steric structural and functional domain, and relative expression's total length, short peptide stretch is easier to vivoexpression, cost-saving, be beneficial to the industrialization of microbial proteinous agricultural chemicals.
Accompanying drawing explanation
Fig. 1 .hrpZ Δ 254-298inverse PCR amplification
Wherein M1 is DNA marker DL10000; M2 is DNA marker DS2000; 1 is hrpZ Δ 254-298.
Fig. 2. recombinant plasmid pET30a (+)-hrpZ Δ 254-298bacterium colony PCR verifies and double digestion checking
The bacterium colony PCR the result of A recombinant plasmid pET30a (+)-hrpZ.Wherein M is DNA marker DS2000; 1 is hrpZ Δ 254-298; 2 is hrpZ psgS1.
The double digestion the result of B recombinant plasmid pET30a (+)-hrpZ.Wherein M1 is DNA marker DL10000; M2 is DNA marker DS2000; 1 is hrpZ Δ 254-298; 2 is hrpZ psgS1.
Fig. 3 .IPTG induces recombinant protein HarpinZ Δ 254-298express and ni-sepharose purification
A IPTG induces recombinant protein HarpinZ Δ 254-298the SDS-PAGE electrophorogram of expressing.Wherein M is protein marker; 1 is HarpinZ Δ 254-298; 2 is HarpinZ psgS1; 3 is pET30.
B HarpinZ Δ 254-298pure protein SDS-PAGE electrophorogram.Wherein M is protein marker; 1 is HarpinZ Δ 254-298; 2 is HarpinZ psgS1.
Fig. 4. recombinant protein HarpinZ Δ 254-298tobacco is induced HR reaction detection
Wherein tandem represents recombinant protein HarpinZ Δ 254-298and contrast HarpinZ psgS1, horizontally-arrangedly represent different volumetric molar concentrations.
Fig. 5. recombinant protein HarpinZ Δ 254-298evoking tobacco opposing mosaic virus (TMV) infects
Upper figure is the scab number schematic diagram that after different albumen process, tobacco mosaic virus (TMV) causes on tobacco leaf.Following table is that after different albumen process, evoking tobacco is to the statistics of TMV disease resisting effect, and X-coordinate is different process, and ordinate zou is evoking tobacco anti-TMV effect per-cent.
Fig. 6. recombinant protein HarpinZ Δ 254-298to the growth-promoting functions of rice paddy seed
A figure is the growth-promoting functions schematic diagram to rice paddy seed after different albumen process, and wherein 1 is recombinant protein HarpinZ Δ 254-298, 2 is total length contrast HarpinZ psgS1, 3 is contrast water.
B figure, C figure are long to rice root after being respectively different albumen process, the statistics of the long growth-promoting effect of seedling, and X-coordinate is different process, and ordinate zou is the length of rice root, seedling.
Fig. 7. recombinant protein HarpinZ Δ 254-298evoking tobacco HCD marker gene hsr515 (A), defense response genes NPR1 (B) expression
Fig. 8. recombinant protein HarpinZ Δ 254-298inducing paddy rice growth related gene OsARF1 expression
Biomaterial preservation information
S1 bacterial strain, Classification And Nomenclature is Pseudomonas syringae pv.glycinea (Pseudomonas syringae pv.glycinea), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC No.6699, address is Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, preservation date is on October 22nd, 2012.
Embodiment
The shortenings used in the present invention:
LB: bacteria culture medium, yeast powder 5g/L+ Tryptones 10g/L+Nacl10g/L, PH7.0;
The kantlex of Km50:50 μ g/ml; IPTG: isopropylthio-beta galactose glycosides; PBS: phosphoric acid buffer
Embodiment 1:hrpZ psgS1the design of gene recombination fragment, choning and sequencing
According to the hrpZ sequence that accession number in Genbank is L41862, design entire open reading frame (ORF) primer, introduces BamH1 and EcoR1 restriction enzyme site respectively at 5 ' end and 3 ' end;
hrpZ PsgS1-P1：5’-GG GGATCCATGCAGAGTCTCAGTCTTAAC-3’(SEQ?ID?No.1)；
hrpZ PsgS1-P2：5’-GG GAATTCTCAGGCAGCAGCCTGGTTG-3’(SEQ?ID?No.2)。
Be template with the genomic dna of S1 bacterial strain (CGMCC No.6699, lower with), carry out pcr amplification hrpZ with above-mentioned primer psgS1full length gene sequence, reaction conditions: 94 DEG C of denaturation 4min, 94 DEG C of sex change 1min, 56 DEG C of annealing 1min, 72 DEG C extend 90s, circulate 35 times, and 72 DEG C extend 10min.PCR primer is connected with pMD18-T carrier, is transformed in bacillus coli DH 5 alpha, obtain positive transformant through plasmid PCR and digestion verification.Extract recombinant plasmid dna and deliver to Nanjing Genscript Biotechnology Co., Ltd.'s order-checking.To recombinant plasmid pMD18-T-hrpZ psgS1sequencing results shows, hrpZ psgS1gene size is 1038bp, 345 amino acid of encoding, and the protein of its coding is rich in glycine (12.75%), not containing halfcystine.
With the DH5 α/pMD18-T-hrpZ built psgS1the plasmid DNA that bacterial strain extracts is that template carries out Inverse PCR amplification, and design of primers is according to the hrpZ of this laboratory clone psgS1sequence (in Genbank, accession number is HM358043), design amplification recombinant protein HarpinZ Δ 254-298the primer of encoding gene:
hrpZ Δ254-298-P1:5’-GGTGGCTTGTTGCAGAAAGGTCTGG-3’(SEQ?ID?No.3)；
hrpZ Δ254-298-P2:5’-CTGGCCCACATCACCATTGGAATTG-3’(SEQ?ID?No.4)。
Reaction conditions: 95 DEG C of denaturation 4min, 98 DEG C of sex change 10s, 57 DEG C of annealing 15s, 72 DEG C extend 4min, circulate 30 times, and 72 DEG C extend 10min.After Inverse PCR amplification success, (Fig. 1) reclaims test kit with gel and amplified fragments is reclaimed purifying, utilize T4DNA ligase enzyme that the fragment after reclaiming is carried out DNA molecular voluntarily after cyclisation, proceed to bacillus coli DH 5 alpha, obtain positive transformant through digestion verification.Extract transformant plasmid DNA and deliver to Nanjing Genscript Biotechnology Co., Ltd.'s order-checking, utilize the BLAST function of NCBI website, measured sequence is compared.Result shows: positive transformant order-checking is correct, obtains the hrpZ that size is 903bp Δ 254-298recombinant fragment (SEQ ID No.5), by positive recombinant bacterial strain called after DH5 α/pMD18T-hrpZ Δ 254-298.
Embodiment 2:HarpinZ Δ 254-298the Expression and purification of recombinant protein
2.1 hrpZ Δ 254-298the structure of gene engineering expression bacterial strain
Check order correct positive recombinant bacterial strain DH5 α/pMD18-T-hrpZ Δ 254-298put forward plasmid DNA, be connected on expression vector pET30a (+) after BamH1 and EcoR1 double digestion, cut (Fig. 2 B) identify positive strain after proceeding to e. coli bl21 through plasmid PCR (Fig. 2 A) and enzyme, order-checking is correct.DH5 α/pMD18-T-hrpZ simultaneously psgS1connect pET30a (+) in the same way, transformation of E. coli BL21, as total length contrast, by these two bacterial strains called after BL21/pET30a (+)-hrpZ respectively Δ 254-298, BL21/pET30a (+)-hrpZ psgS1.
2.2 HarpinZ Δ 254-298the expression of recombinant protein
(1) IPTG induces expression of recombinant proteins: by recombinant bacterial strain BL21/pET30a (+)-hrpZ Δ 254-298, BL21/pET30a (+)-hrpZ psgS1be inoculated in 37 DEG C of (200rpm/min) shaking culture in LB liquid nutrient medium (km50 μ g/ml) respectively to spend the night, next day according to 1% ratio be transferred in the liquid LB of km50,37 DEG C of shaking culture are to OD 600=0.6 ~ 0.8, add the IPTG that final concentration is 0.1mM, 28 DEG C of shaking culture 3-4h.
(2) ultrasonic disruption obtains thick leach protein: collect bacterium liquid 12000rpm/min, the centrifugal 10min of normal temperature collects thalline, heavily be dissolved in the PBS of 1/20 former yeast culture volume, fragmentation is carried out with sonicator after suspension thalline, 12000rpm/min after broken, 4 DEG C of centrifugal 10min, obtain supernatant liquor and are the thick leach protein of HarpinZ.SDS-PAGE detects HarpinZ Δ 254-298albumen size is 37.4KD, HarpinZ psgS1albumen size is 42kD (Fig. 3 A).
2.3 HarpinZ Δ 254-298the purifying of recombinant protein
Use HisTrap tMhP (GE Healthcare) prepacked column carries out purifying to recombinant protein, and concrete steps are with reference to specification sheets.Before purifying, with 0.22 μm of thick leach protein of membrane filtration harpinZ, remove some cell debriss or other impurity in thick leach protein, with antifouling purification column, during purifying, use peristaltic pump loading.Elutriant after purifying carries out ultrafiltration, concentrated through the super filter tube that the molecular weight cut-off of Millipore company is 30KD, finally obtains single pure protein band (Fig. 3 B).Utilize BSA determination of protein concentration kit measurement protein concentration, pure protein is in-70 DEG C of preservations.
Embodiment 3:HarpinZ Δ 254-298the application of recombinant protein on plant
3.1 HarpinZ Δ 254-298recombinant protein evoking tobacco HR reacts
Tobacco detects recombinant protein HarpinZ Δ 254-298induction produces the ability of HR, simultaneously with HarpinZ psgS1be treated to contrast with clear water, inject 200 μ l with syringe without a head to intercellular substance, the tobacco leaf back side, pure protein concentration used is 10nmol/ml, 7nmol/ml, 2.5nmol/ml, 1.5nmol/ml, 1.2nmol/ml, observes blade HR development after 24h.
Result shows recombinant protein HarpinZ Δ 254-298though eliminate the Partial Fragment of total length, but still can on tobacco, induce HR to react, just evoking tobacco HR ability comparatively total length compare and weaken, minimum concentration is 7 μm/ml (Fig. 4).
3.2 HarpinZ Δ 254-298recombinant protein effectively improves the resistance of tobacco to mosaic virus
Use final concentration is 7nmol/ml pure protein HarpinZ Δ 254-298the tobacco leaf of spraying process in advance, with the HarpinZ of same concentration psgS1, clear water spraying be treated to contrast.Frictional inoculation tobacco mosaic virus (TMV) after 12h, after 3 days, observes blade incidence, statistics scab number.
Result shows, HarpinZ Δ 254-298, HarpinZ psgS1after process tobacco leaf, occurring degree significantly alleviates compared with clear water process, and HarpinZ Δ 254-298the anti-TMV effect of evoking tobacco of albumen reaches 96%, is significantly higher than total length contrast HarpinZ psgS171.6% (Fig. 5), the above results shows, the HarpinZ that we obtain Δ 254-298recombinant protein has better mosaic disease resisting toxic effect fruit, is significantly higher than total length HarpinZ psgS1.
3.3 HarpinZ Δ 254-298recombinant protein promotes young rice seedlings growth
Same use final concentration is 7nmol/ml pure protein HarpinZ Δ 254-298rice paddy seed after soaking disinfection, with the HarpinZ of same concentration psgS1, clear water is treated to contrast.Sucking-off protein liquid after 12h, aqua sterilisa is inverted on the filter paper of sterilizing after cleaning three times, blotting after water until seed is wrapped in square filter paper, often open filter paper and wrap up 8 pieces of seeds, filter paper volume is vertically put into the plastic cup filling clear water, the water surface is no more than seed position, is placed in illumination box and cultivates, and each process arranges 10 repetitions.The rice root of 10 days statistics different treatment is long, seedling is long, and application SPSS software carries out statistical study to data.
Statistics shows, HarpinZ Δ 254-298, HarpinZ psgS1compared with water, seedling length long to the root of paddy rice has obvious growth promoting function, and recombinant protein HarpinZ Δ 254-298to the growth-promoting functions of paddy rice apparently higher than HarpinZ psgS1total length contrasts, and there is significant difference (Fig. 6) between the two.This result shows, the HarpinZ that we obtain Δ 254-298recombinant protein in short paddy growth higher than total length HarpinZ psgS1.
4.HarpinZ Δ 254-298the expression of recombinant protein inducing plant body inside defense reacting phase correlation gene, growth related gene
On tobacco, use final concentration is 7nmol/ml pure protein HarpinZ Δ 254-298spraying process blade, with the HarpinZ of same concentration psgS1, clear water spraying is treated to contrast, in 0h, 3h, 6h, 9h, 12h, 18h, 24h, sample respectively, extract leaf portion RNA, detected the expression of anaphylaxis genes involved hsr515 in tobacco, Defense response gene NPR1 by Real-time PCR.Result shows HarpinZ Δ 254-298, HarpinZ psgS1the up-regulated expression of hsr515, NPR1 gene can be caused, and HarpinZ Δ 254-298hsr515 gene expression amount contrast HarpinZ lower than total length psgS1, and NPR1 gene expression amount contrasts HarpinZ apparently higher than total length psgS1(Fig. 7), this shows that expression conditions conforms to the Phenotypic examination result on tobacco.
On paddy rice, final concentration is 7nmol/ml pure protein HarpinZ Δ 254-298spraying process growth 30d rice leaf, with the HarpinZ of same concentration psgS1, clear water spraying is treated to contrast, sample after 6h, extract leaf portion RNA, detected the expression of growth related gene OsARF1 by Real-time PCR.Result shows HarpinZ Δ 254-298, HarpinZ psgS1the up-regulated expression of OsARF1 gene can be caused, and HarpinZ Δ 254-298expression amount contrast HarpinZ apparently higher than total length psgS1(Fig. 8), HarpinZ is demonstrated from gene level Δ 254-298promote the biological effect of paddy growth.
Above example shows the HarpinZ that the present invention obtains psgS1recombinant protein HarpinZ after brachymemma Δ 254-298plant can excite a series of useful biological effect.HarpinZ Δ 254-298though eliminate Partial Fragment, but still anaphylaxis can be excited on non-host plant tobacco; The resistance of tobacco to mosaic virus can be significantly improved, higher than total length HarpinZ after process tobacco psgS1; Long to the root of paddy rice after Rice seeds treated, seedling length has obvious growth promoting function, higher than total length HarpinZ psgS1; And these useful effects on plant all obtain checking from gene level.

Claims (2)

1. the recombinant protein HarpinZ of aminoacid sequence as shown in SEQ ID NO.6 Δ 254-298improving the application in Resistance In Tobacco tobacco mosaic virus disease effect, promotion paddy growth.
2. application according to claim 1, is characterized in that described recombinant protein HarpinZ Δ 254-298prepared by following steps:
(1) with DH5 α/pMD18-T-hrpZ psgS1the plasmid DNA that bacterial strain extracts is template, uses upstream primer hrpZ Δ 254-298-P1, downstream primer hrpZ Δ 254-298-P2 carries out Inverse PCR amplification, reclaim test kit with gel and the fragment of Inverse PCR amplification is reclaimed purifying, utilize T4DNA ligase enzyme that the fragment after reclaiming is carried out DNA molecular voluntarily after cyclisation, proceed to bacillus coli DH 5 alpha, to cut through enzyme and sequence verification obtains positive recombinant bacterial strain DH5 α/pMD18T-hrpZ Δ 254-298, wherein upstream primer hrpZ Δ 254-298-P1 sequence as shown in SEQ ID NO.3, downstream primer hrpZ Δ 254-298-P2 sequence is as shown in SEQ ID NO.4;
(2) positive recombinant bacterial strain DH5 α/pMD18-T-hrpZ Δ 254-298put forward plasmid DNA, be connected to after BamH1 and EcoR1 double digestion on expression vector pET30a (+), cut through plasmid PCR and enzyme after proceeding to e. coli bl21 and identify positive strain BL21/pET30a (+)-hrpZ Δ 254-298;
(3) LB culture medium culturing positive strain BL21/pET30a (+)-hrpZ is utilized Δ 254-298, and utilizing IPTG to induce expression of recombinant proteins, thalline obtains thick leach protein through ultrasonic disruption, obtains the recombinant protein HarpinZ of purifying after purifying Δ 254-298.
CN201410510546.1A 2014-09-28 2014-09-28 Application of recombinant protein HarpinZDelta254-298 Pending CN104311646A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410510546.1A CN104311646A (en) 2014-09-28 2014-09-28 Application of recombinant protein HarpinZDelta254-298

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410510546.1A CN104311646A (en) 2014-09-28 2014-09-28 Application of recombinant protein HarpinZDelta254-298

Publications (1)

Publication Number Publication Date
CN104311646A true CN104311646A (en) 2015-01-28

Family

ID=52366982

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410510546.1A Pending CN104311646A (en) 2014-09-28 2014-09-28 Application of recombinant protein HarpinZDelta254-298

Country Status (1)

Country Link
CN (1) CN104311646A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108264563A (en) * 2016-12-30 2018-07-10 四川本原作物科技有限公司 Covalent trimeric polypeptides of glucan-modified Hrps and preparation method thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103103202A (en) * 2013-01-16 2013-05-15 南京农业大学 Harpin coding gene (i) hrpZPsgS1(/i) or application of harpinZPsgS1 protein expressed by Harpin coding gene (i) hrpZPsgS1(/i)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103103202A (en) * 2013-01-16 2013-05-15 南京农业大学 Harpin coding gene (i) hrpZPsgS1(/i) or application of harpinZPsgS1 protein expressed by Harpin coding gene (i) hrpZPsgS1(/i)

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
CHUN-MEI LI: "Harpin of Pseudomonas syringae pv. phaseolicola Harbors a Protein Binding Site", 《MPMI》 *
K. ANIL: "LEUCINE ZIPPER-LIKE MOTIFS OF HRPZPSS ARE NOT ESSENTIAL TO INDUCE HYPERSENSITIVE RESPONSE IN TOBACCO", 《JOURNAL OF PLANT PATHOLOGY》 *
MINNA HAAPALAINEN: "Functional mapping of harpin HrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization, lipid interactions and plant defence induction", 《MOLECULAR PLANT PATHOLOGY》 *
MIN-SEON CHOI: "Harpins, Multifunctional Proteins Secreted by Gram-Negative Plant-Pathogenic Bacteria", 《MPMI》 *
张岩: "丁香假单胞大豆致病变种harpin 编码基因的克隆表达与功能研究", 《南京农业大学学报》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108264563A (en) * 2016-12-30 2018-07-10 四川本原作物科技有限公司 Covalent trimeric polypeptides of glucan-modified Hrps and preparation method thereof

Similar Documents

Publication Publication Date Title
CN102010466B (en) Plant resistance associated protein MYB, as well as coding gene and application thereof
CN103103202A (en) Harpin coding gene (i) hrpZPsgS1(/i) or application of harpinZPsgS1 protein expressed by Harpin coding gene (i) hrpZPsgS1(/i)
CN102675434B (en) Magnaporthe oryzae isolated protein for improving plant resistance and inducing defense reaction of plant and gene and application of magnaporthe oryzae isolated protein
CN108314714A (en) Verticillium dahliae secreted protein exciton VdPEL1 and its application
CN102187874B (en) Application of pathogenic gene related to xanthomonas campestris pathovar campestris
CN101153057B (en) Protein for improving plants fastness and accelerating plant growth and encoding gene thereof
CN108070605B (en) Carbendazim degrading enzyme CbmA as well as coding gene and application thereof
CN104322530B (en) Recombinant protein HarpinZ Δ 89-124application
CN104311646A (en) Application of recombinant protein HarpinZDelta254-298
CN102659933B (en) Bacillus thuringiensis gene cry8like and cry8G combination and application thereof
CN101979503B (en) Recombinant brucella for expressing Asia type-I foot and mouth disease virus VP1 genes and method for producing vaccines thereof
CN104862331B (en) A kind of method of solubility expression Rhodococcus equi Disease-causing gene VapA albumen
CN104277114B (en) A kind of new fusion protein and the legionella pneumophilia vaccine with its preparation
CN103254301B (en) Plant disease resistance related protein GbMBL1, its coding gene and application
CN103665126A (en) HbsHsp2 protein coming from hevea brasiliensis, and encoding gene and application thereof
CN103290000B (en) SCAR marker of biocontrol Hypocrea virens, its application and quantitative detection method
CN102154315A (en) Ipomoea batatas metallothionein gene (IbMT1) and application thereof in improving salt resistance and drought resistance of plants
CN109134629A (en) Botrytis cinerea secreted protein exciton BcXyl1 and its application
CN107502552A (en) A kind of green fluorescence fusarium prolifertum and its application
CN111139207A (en) Brevibacillus brevis gene recombinant strain and preparation method and application thereof
CN114306574B (en) Koi protein for resisting pathogenic bacteria infection and application thereof
CN103484477B (en) Latcripin-15 gene segment of mushroom C91-3 strain, coded protein and preparation method
CN103205439B (en) Pseudomonas syringae pv. glycinea harpinZ psgA1the expression of albumen and application
CN103589735A (en) Solanum torvum StoWRKY6 gene and expression vector and applications thereof
CN103060333A (en) Litopenaeus vannamei metallothionein gene LvMT as well as coding gene and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20150128