CN1888052A - Separation and purification of protein BS2 resisting cotton blight and verticillium wilt and cloning of BS2 gene - Google Patents
Separation and purification of protein BS2 resisting cotton blight and verticillium wilt and cloning of BS2 gene Download PDFInfo
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Abstract
The present invention is one kind of BS2 protein separated and purified from Bacillus subtilis B111 fermenting liquid and capable of resisting cotton blight and verticillum wilt. The initial functional identification shows that the BS2 protein possesses certain inhibition on cotton blight and verticillum wilt and E.coli. Identification shows that the BS2 protein belongs to neutral proteinase of Bacillus subtilis, and the gene sequence of the BS2 protein has been cloned from Bacillus subtilis B111. The BS2 protein may be used in the development of biological preparation for preventing and controlling cotton blight and verticillum wilt, and the gene may be used in the research of blight and verticillum wilt resisting transgenic cotton.
Description
Technical field
The present invention relates to the wither subtilis B111 of disease, verticillium of a kind of resisting cotton blight, derive from BS2 albumen and the relevant BS2 gene order thereof of this bacterium.This bacterium and BS2 albumen thereof have certain restraining effect to the growth of verticillium dahliae and blight.This BS2 albumen can be used for the research of biological pesticide or biological prevention and control agent.This BS2 gene can be used for transgene cotton disease resistance expression study.
Background technology
Plant diseases is a world food underproduction one of the main reasons, cause 10%～15% production loss every year in farm crop produce, the cotton loss is about 12% (Hainr and P.H.Schireier, Journal of.Pflanzenschutz-Nachrichten Bayer, (special issue), 49 (67): 25-120,1996).Wherein fungal diseases of plants is positioned at plant four big diseases (fungi, bacterium, virus and nematode (Zheng Xiufang etc., Gansu science journal, 13 (1): 63-66,2002)), account for more than 80% of Plant diseases sum (Luo Yanliang etc., biology magazine, (6): 6-8,1994).And the blight and the verticillium that are called as cotton two big " cancers " are one of most important diseases of Cotton Production.It is one of main disease of cotton that Fusarium oxysporum Schl.f.sp.vasinfectum (Fusariumoxysporum f.sp.vasinfectum) infects the cotton wilt that causes, output of cotton and quality are caused very big influence.The cotton verticillium wilt (Verticillium dahliaeKleb) that is caused after contaminating by black and white Verticillium, big beautiful Verticillium is the another important cotton disease that it is found that after blight simultaneously, is distributed widely in the world and respectively produces cotton state.The Cotton in China verticillium was imported into by introducing the american cotton kind from nineteen thirty-five, over nearly 10 years, cotton verticillium wilt is on the rise, the grave illness ground cotton underproduction 60%～70%, that have even total crop failure, seriously having hindered the development that Cotton in China produces, (equality is defended in the room, the Henan agricultural sciences, (1): 7-9,1998; Li Peifu, the land-reclaimable science and technology in Xinjiang, (2): 12-13,1998).Particularly occurred being similar to the pathogenic by force verticillium wilt pathogen of U.S. T9 defoliation in recent years, even more serious to the harm of Cotton in China verticillium, not only epidemic regions constantly enlarges, and loss is more serious.Cotton defoliation verticillium becomes influences Cotton in China acquisition one of high yield, the major obstacle of stable yields and subject matter of Cotton Production Sustainable development after insect pest.Therefore, the anti-defoliation verticillium of seed selection, blight cotton variety as early as possible, control spreads, stops the variation that delays germ, prevents that blight, verticillium are popular on a large scale, avoid the loss to bringing on a disaster property of Cotton in China production.
The present invention is separated to a strain bacterium, called after B111 from the Chinese medicine Fructus Amomi Rotundus.Dull and stereotyped antagonistic experiment result shows the growth of this bacterium energy strongly inhibited verticillium and blight.Determine that by physiological and biochemical test and 16S rRNA qualification result B111 belongs to subtilis.Cell free fermentation liquid to this bacterium carries out separation and purification, and in conjunction with dull and stereotyped antagonistic effect, finds that a kind of albumen of this bacterium excretory (called after BS2) all has restraining effect to the growth of defoliation verticillium wilt pathogen V991 and E.coli, does not have influence to pichia spp.Growth to blight Ag226 simultaneously also has certain restraining effect.Utilize online anti-phase capillary column liquid chromatography esi-msn CapLC-ESI-MS-MS to obtain proteic five amino acid sequence of polypeptide of BS2, second order ms database analysis result is presented at this albumen and genus bacillus neutral protease (BacillolysinEC 3.4.24.28) homology is higher, may be a kind of new genus bacillus neutral protease.According to survey amino acid sequencing result and homologous sequence design primer thereof, obtained the complete genome sequence of BS2 albumen 1.9kb.
Also fungi do not had reporting for work of resistance at present by retrieval about the subtilis neutral protease.But people such as Chen Yun (Chen Yun etc., functional polymer journal, 12 (3): 293-296,1999) have reported the neutral protease nonspecific hydrolyzing chitosan of energy and its reaction conditions have been studied.Chitosan extensively is present in the fungi, is the important component part of fungal cell wall.This may be exactly one of antibacterial protein BS2 reason that can suppress verticillium wilt pathogen growth.
Summary of the invention
The invention provides a kind of bacterial strain of preventing and treating cotton wilt, verticillium: Bacillussubtilis B111 (depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica; Deposit number: CGMMCC No.1497; Preservation date: on October 18th, 2005; Classification name: subtilis Bacillus subtilis).
Another object of the present invention provides a kind of that derive from subtilis B111 and cotton wilt, gene that verticillium is relevant: the BS2 gene.
BS2 gene provided by the invention is one of following nucleic acid row:
1) the SEQ ID NO:1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of SEQ ID NO:1 have 90% above homology, and encode and have the wither protein DNA sequence of disease, verticillium function of resisting cotton blight.
SEQ ID NO:1 in the sequence table is by 1908 based compositions.The reading frame of this gene is that SEQ ID NO:1 holds the 254th to the 1819th bit base from 5 '.
A further object of the present invention provides a kind of by the wither protein with antibacterial of the relevant BS2 genes encoding of disease, verticillium of resisting cotton blight, be the amino acid residue sequence of SEQ ID NO:2 in the sequence table, or the amino acid residue sequence of SEQ ID NO:2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence SEQ ID NO:2 deutero-protein with the amino acid residue sequence of SEQ ID NO:2.
The protein that SEQ ID NO:2 in the sequence table is made up of 521 amino acid.
Contain expression carrier of the present invention and clone and all belong to protection scope of the present invention.
BS2 gene of the present invention can be imported in the plant such as cotton, in the hope of obtaining transgene cotton that blight, verticillium are had resistance.Gene pairs of the present invention is cultivated the wither plant variety of disease, verticillium of adversity resistant plant kind, particularly resisting cotton blight, and it is significant to improve crop yield and quality.
The present invention will be further described below in conjunction with accompanying drawing
The explanation of accompanying drawing table
Fig. 1: QAE sephadex A50 reinforcing yin essence ion exchange column elution curve.
Fig. 2: SDS-PAGE protein electrophoresis figure.1,2,3,4 represent the protein crude extract of B111, the fermenation raw liquid of B111 respectively among the figure, and elution peak 1 is collected liquid, elution peak 2 (being BS2 albumen) is collected liquid.
Fig. 3: Oxford cup bacteriostatic experiment figure.(A): subtilis B111 is to the restraining effect of defoliation verticillium V991; (B): with the restraining effect of the thick leach protein after the 90% ammonium sulfate precipitation B111 fermented liquid to verticillium V991; (C) elution peak 1 is collected the restraining effect of liquid to verticillium V991; (D) elution peak 2 (being BS2) is collected the restraining effect of liquid to verticillium V991.
Fig. 4: the proteic molecular weight of BS2 that utilizes MALDI-TOF-MS to measure.
Fig. 5: the proteic peptide finger printing of BS2 (PFM) that adopts MALDI-TOF-MS to measure.
Fig. 6: metal ion activation experiment figure.(A) zincification ionic fungistatic effect not in the BS2 albumen damping fluid (20mmol/LTris-HCl, pH 7.0); (B) add (20mmol/L Tris-HCl, 2mmol/L Zn behind the zine ion in the BS2 albumen damping fluid
2+, pH 7.0) fungistatic effect.
Fig. 7: according to amino acid sequencing result and second order ms database search consequence devised primer thereof, the nucleotide sequence (swimming lane 1) of the 729bp of the BS2 albumen correspondence that increased.
Fig. 8:, obtained the full length DNA sequence 1908bp (swimming lane 1) of BS2 gene according to homologous sequence design primer.
Embodiment
The separation and purification of embodiment 1 antibacterial protein BS2
The cultivation of 1 subtilis B111 and the processing of fermented liquid
Subtilis B111 is inoculated into LB substratum (the yeast powder 5g/l of sterilization, acid hydrolysis casein 25g/l, NaCl10g/l, pH7.0) in, 37 ℃, 250rmp, after cultivating 24h, antagonism bacterium B111 fermented liquid is placed centrifugal bottle, centrifugal 20 minutes of 4 ℃, 12000rpm, abandon or adopt precipitation, keep supernatant.
Separation, the purifying of body I2 antibacterial protein BS2
Employing molecular interception amount is that the ultra-fine filter of 20KD concentrates subtilis B111 fermented liquid supernatant.Take by weighing a certain amount of ammonium sulfate (the salt final concentration reaches 90%) according to the volume that concentrates secondary fermentation liquid, before adding, ammonium sulfate must be ground into powdery with mortar, under 4 ℃, slowly join in the concentrated broth after centrifugal, the limit edged stirs, slowly carry out, avoid partial concn too high, cause protein precipitation inhomogeneous.Stir after 2 hours, be placed on to precipitate in 4 ℃ of refrigerators and spend the night.In centrifugal 40 minutes of 4 ℃, 15000rpm, keep precipitation with crude extract next day, abandons or adopts supernatant.Use a small amount of damping fluid (20mmol/L Tris-HCl, pH 7.0) dissolution precipitation then, place 24h in the dialysis band (10mm, MWCO 1000Da) again, carry out desalting treatment (, using same damping fluid dialysis again instead) earlier with pure water dialysis 2 hours.After dialysis finishes, get 20ul and carry out electrophoresis checking extraction effect, and get and carry out bacteriostatic experiment in right amount.Adopt reinforcing yin essence ion exchange column (filler: QAEsephadexA50; Column type: 0.9 * 30cm; Elutriant: solution A: 20mmol/L Tris-HCl, pH 7.0, solution B: 1mol/LNaCl, pH7.0) protein crude extract is carried out continuous gradient wash-out (pH is constant, increases the elutriant ionic strength), elution flow rate is 1ml/min, the detection wavelength is 280nm.Collect the elutriant of each elution peak respectively, the attention collection process is carried out under 4 ℃ of conditions.Obtain two elution peaks (Fig. 1) altogether through gradient elution, the elutriant of each elution peak is placed 24h in the dialysis band (10mm, MWCO 1000Da) respectively, carry out desalting treatment (with pure water dialysis 2 hours, use instead again among the same damping fluid ie in solution A and dialyse earlier).Adopt PEG20000 that the liquid in each dialysis band (10mm, MWCO 1000Da) is concentrated at last.The employing separation gel is 15% SDS-PAGE electrophoresis detection purity of protein (Fig. 2).
The cultivation of 1 defoliation verticillium dahliae V991:
Get the sterilized water that V991 on an amount of inclined-plane places 200ul, make spore suspension and be evenly coated on Czapek ' the s plate culture medium, cultivated 5 days at 28 ℃.Again the 10ml sterilized water is added on the flat board, behind the mixing this high density spore suspension is added in the 100ml sterilized water, make OD
595=0.795 spore suspension, standby by being stored in 4 ℃ after every pipe 1ml packing.
The detection of 2 bacteriostatic activities
Get the V991 spore suspension that 200 μ l prepare and evenly spread upon Czapek ' s (KNO
31.36g/L, NaCl 1.38g/L, KH
2PO
4Lg/L, FeSO
40.02g/L, sucrose 30g/L, agar powder 15g/L) and in the dull and stereotyped culture dish (diameter 90cm), and at dull and stereotyped moderate distance placement Oxford cup, add 100 μ l protein samples in each Oxford cup, control group then adds 100 μ l damping fluids (20mmol/L Tris-HCl, pH 7.0).Place 28 ℃ of cultivations, take out the Oxford cup after second day absorption of sample, continue to cultivate the growing state (Fig. 3) of observing the verticillium dahliae that is added with protein example and contrast in 4 days at 28 ℃.The collection liquid of bacteriostatic test proof elution peak 2 can suppress the growth of defoliation verticillium V991, and SDS-PAGE electrophoresis detection result shows that elution peak 2 is single albumen, and molecular weight is about 36kD.Therefore think that tentatively this albumen is antibacterial albumen, called after BS2.
The evaluation of embodiment 3 antibacterial protein BS2
Utilize MALDI-TOF-MS to measure proteic molecular weight of BS2 and peptide finger printing (PFM) (Fig. 4,5) thereof.The BS2 molecular weight of measuring is 32048.979Da.Adopt online anti-phase capillary column liquid chromatography esi-msn CapLC-ESI-MS-MS to obtain proteic five amino acid sequence of polypeptide of BS2 (table 1) simultaneously, utilize Mascot to obtain significant search in the second order ms storehouse, the result shows that this albumen and two protein (hydrolyzed starch genus bacillus neutral protein enzyme precursor HYBSN gi|67706 and genus bacillus B16 neutral protein enzyme precursor bael6 AAV30844 gi|54126578) have homology
These two homologous proteins all belong to the genus bacillus neutral protease, and between these two homologous proteins homology up to 89%.5 measured amino acid sequence of polypeptide all are positioned at the maturation protein zone of homologous protein simultaneously.In addition, the BS2 molecular weight of albumen also conforms to substantially with the maturation protein molecular weight of this series bacillus neutral protease, all is about 32-33kD.Therefore infer that BS2 may be a kind of new genus bacillus neutral protease.
The comparison of table 1BS2 sequencing result and homologous sequence
| NO. | BS2 | HYBSN | AAV30844 | |
| 1 2 3 4 5 | TVSLNISSENGK AAVDAHYNLGK YGQPDHYK NYKNLPNTDAGD YGGVHTNSGIPNK KAEQIYYR | TVSLNISSESGK AAVDAHYNLGK YGQPDNFK NYKNLPNTDAGD YGGVHTNSGIPNK KAEQIYYR | TVSLNISSESGK AAVDAHYNLGK YGQPDHYK NYRNLPNTDAGD YGGVHTNSGIPNK KAEQIYYH | 235-246 291-301 425-432 433-457 470-477 |
The activation of embodiment 4 antibacterial protein BS2 bacteriostatic activities
Property analysis to the genus bacillus neutral protease is found (Bacillolysin EC 3.4.24.28), this fermentoid is a metalloprotease, it has two metal ion species binding sites i.e. three zine ions and a calcium ion binding site, and its response characteristic is similar to thermolysin.And zine ion is relevant with the enzymic activity of thermolysin, and calcium ion is relevant with the stability of enzyme.Therefore may cause enzymic activity to decrease owing to lost essential metal ion in the purge process than protein crude extract administration.Thereby in the albumen damping fluid, added zine ion and calcium ion respectively, and make the metal ion final concentration reach 2mmol/L, carry out the bacteriostatic test test again.Test-results shows that calcium ion does not have obvious activation to BS2 albumen bacteriostatic activity, and zine ion has certain activation (Fig. 6) to this proteic bacteriostatic activity.
The clone of embodiment 5BS2 gene
1 chooses two sections sequences (TVSLNISSE and KAEQIYYR) of the most close N-end and C-end as special primer from order-checking gained aminoacid sequence, design primers F 1:5 '-ACGG TCTCATTAAATAATTCTTCTGAA-3 ', R1:5 '-ACGATAGTAAATCTGCTCCGCTTT-3 ', with the B111 genomic dna is that template is carried out PCR (Fig. 7), has obtained the nucleic acid fragment about about 700bp.To last amino acid R (477) of primer 2 coding, should comprise 243 amino acid prefaces from first amino acid T (235) of primers F 1 coding, corresponding DNA sequences should be 729bp. and PCR result is checked order shows and to fulfill the expectation that this fragment length is 729bp.This sequence is translated into aminoacid sequence according to bacterium preference codon, and the theoretical restriction enzyme mapping of its aminoacid sequence and BS2 peptide finger printing PMF are identical, and then think the partial sequence that obtains antibiotic protein gene.
2 after this according to known homologous sequence hydrolyzed starch genus bacillus neutral protein enzyme precursor HYBSN gi|67706 design homology primer, F2:5 '-GATCTTAACATTTTTCCCCTATCATTT---3 '; R2:5 '-CGGGAA AAGACATATATCATCATGGT-3 ', the full length sequence 1908bp (Fig. 8) of this proteic BS2 gene that obtained to encode.The open reading frame of this sequence is held the 254th to the 1819th bit base from 5 '.521 amino acid of encoding altogether are signal peptide with the more measurable 1-27 of a homologous sequence amino acid wherein, 28-221 amino acids position precursor peptide, and the 222-521 amino acids is the genus bacillus neutral protease for the maturation protein zone.
Claims (10)
1, a kind of bacterial strain of preventing and treating cotton wilt, verticillium is characterized in that this bacterial classification is subtilis (Bacillussubtilis) B111 bacterial strain CGMMCC No.1497.
2, a kind of anti-blight of subtilis B111, BS2 gene of verticillium wilt pathogen of deriving from is one of following nucleotide sequence:
1) the SEQ ID NO:1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of SEQ ID NO:1 have 90% above homology, and encode and have the wither protein DNA sequence of disease, verticillium function of resisting cotton blight.
3, a kind of anti-blight of subtilis B111, BS2 albumen of verticillium wilt pathogen of deriving from is one of aminoacid sequence:
1) the SEQ ID NO:2 in the sequence table;
2) with sequence table in the aminoacid sequence that limits of SEQ ID NO:2 have 90% above homology, and have the wither proteinic aminoacid sequence of disease, verticillium function of resisting cotton blight.
4, the BS2 gene of anti-blight according to claim 2, verticillium wilt pathogen is characterized in that: described gene is the SEQ ID NO:1 in the sequence table.
5, the BS2 gene of anti-blight according to claim 2, verticillium wilt pathogen is characterized in that: described gene is the proteic dna sequence dna of BS2 of coding subtilis B111 excretory anti-blight, verticillium.
6, gene according to claim 2, it is characterized in that: have the protein of the amino acid residue sequence of SEQ ID NO:2 in the sequence table, or the amino acid residue sequence of SEQ ID NO:2 is passed through replacement, disappearance or the interpolation of one or several amino-acid residue and has identical active by sequence SEQ ID NO:2 deutero-protein with the amino acid residue sequence of SEQ ID NO:2.
7, gene according to claim 2 is characterized in that: the reading frame of this gene is for holding the 254th to the 1819th bit base from 5 '.
8, contain the described expression carrier of claim 1.
9, the clone that contains the described gene of claim 1.
10, the application of the described gene of claim 1 in cultivating the disease-resistant plants kind.
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Cited By (5)
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| CN101671633B (en) * | 2009-09-28 | 2010-11-10 | 南京农业大学 | Antagonistic bacteria for preventing and eliminating greensickness of continuous cropping cotton and microbial organic fertilizer thereof |
| CN101928338A (en) * | 2010-09-03 | 2010-12-29 | 河北农业大学 | Cotton GbVe gene, protein coded thereby and use thereof in vegetable verticillium wilt resistance |
| CN102321162A (en) * | 2011-10-08 | 2012-01-18 | 左开井 | GbRPS1 gene for resisting plant fusarium wilt and verticillium wilt and application thereof |
| CN103125989A (en) * | 2012-03-31 | 2013-06-05 | 大连工业大学 | Method of extracting fish cerebrol and fish brain peptide |
| CN112813086A (en) * | 2019-11-15 | 2021-05-18 | 中国农业科学院生物技术研究所 | Gene with verticillium wilt resistance function and application thereof |
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| CN1304658A (en) * | 2001-01-11 | 2001-07-25 | 崔西会 | Disinfectant |
| CN1335073A (en) * | 2001-09-05 | 2002-02-13 | 罗萌 | Ecological pesticide of plant material and its compounding process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671633B (en) * | 2009-09-28 | 2010-11-10 | 南京农业大学 | Antagonistic bacteria for preventing and eliminating greensickness of continuous cropping cotton and microbial organic fertilizer thereof |
| CN101928338A (en) * | 2010-09-03 | 2010-12-29 | 河北农业大学 | Cotton GbVe gene, protein coded thereby and use thereof in vegetable verticillium wilt resistance |
| CN101928338B (en) * | 2010-09-03 | 2012-12-26 | 河北农业大学 | Cotton GbVe gene, protein coded thereby and use thereof in vegetable verticillium wilt resistance |
| CN102321162A (en) * | 2011-10-08 | 2012-01-18 | 左开井 | GbRPS1 gene for resisting plant fusarium wilt and verticillium wilt and application thereof |
| CN102321162B (en) * | 2011-10-08 | 2013-05-08 | 左开井 | GbRPS1 gene for resisting plant fusarium wilt and verticillium wilt and application thereof |
| CN103125989A (en) * | 2012-03-31 | 2013-06-05 | 大连工业大学 | Method of extracting fish cerebrol and fish brain peptide |
| CN103125989B (en) * | 2012-03-31 | 2014-08-06 | 大连工业大学 | Method of extracting fish cerebrol and fish brain peptide |
| CN112813086A (en) * | 2019-11-15 | 2021-05-18 | 中国农业科学院生物技术研究所 | Gene with verticillium wilt resistance function and application thereof |
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