Separation and purification of protein BS2 resisting cotton blight and verticillium wilt and cloning of BS2 gene

A cotton fusarium wilt and fusarium wilt resistance technology, applied in the direction of genetic engineering, plant genetic improvement, chemical instruments and methods, etc., to achieve the effect of improving crop yield and quality

Inactive Publication Date: 2007-01-03
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI +1
View PDF0 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] After searching, there is no report about the resistance of Bacillus subtilis neutral protease to fungi

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Separation and purification of protein BS2 resisting cotton blight and verticillium wilt and cloning of BS2 gene
  • Separation and purification of protein BS2 resisting cotton blight and verticillium wilt and cloning of BS2 gene
  • Separation and purification of protein BS2 resisting cotton blight and verticillium wilt and cloning of BS2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The separation and purification of embodiment 1 antimicrobial protein BS2

[0026] 1 Cultivation of Bacillus subtilis B111 and treatment of fermentation broth

[0027] Inoculate Bacillus subtilis B111 into sterilized LB medium (yeast powder 5g / l, acid hydrolyzed casein 25g / l, NaCl10g / l, pH 7.0), culture at 37°C, 250rmp for 24h, and the antagonistic bacteria Place the B111 fermentation broth in a centrifuge bottle, centrifuge at 12,000 rpm for 20 minutes at 4°C, discard the precipitate, and keep the supernatant.

[0028] Isolation and Purification of Body I2 Antibacterial Protein BS2

[0029] The supernatant of the Bacillus subtilis B111 fermentation broth was concentrated by using an ultrafilter with a molecular cutoff of 20KD. Weigh a certain amount of ammonium sulfate according to the volume of the fermented liquid after concentration (the final concentration of salt reaches 90%). Before adding, the ammonium sulfate must be ground into powder with a mortar, and slow...

Embodiment 2

[0030]Embodiment 2 antibacterial activity identification

[0031] 1 Cultivation of deciduous cotton Verticillium dahliae V991:

[0032] Take an appropriate amount of V991 on the slant and place it in 200ul of sterile water to make a spore suspension and spread it evenly on the Czapek's plate medium, and culture it at 28°C for 5 days. Add 10ml of sterile water to the plate, mix well and add the high concentration spore suspension to 100ml of sterile water to make OD 595 =0.795 spore suspension, divided into 1ml per tube and stored at 4°C for later use.

[0033] 2 Detection of antibacterial activity

[0034] Take 200 μl prepared V991 spore suspension and spread evenly on Czapek’s (KNO 3 1.36g / L, NaCl 1.38g / L, KH 2 PO 4 lg / L, FeSO 4 0.02g / L, sucrose 30g / L, agar powder 15g / L) plate culture dish (diameter 90cm), and Oxford cups were placed equidistantly in the plate, 100μl protein sample was added to each Oxford cup, and 100μl buffer was added to the control group. solution ...

Embodiment 3

[0035] Identification of embodiment 3 antibacterial protein BS2

[0036] The molecular weight of BS2 protein and its peptide fingerprint (PFM) were determined by MALDI-TOF-MS ( Figure 4 , 5 ). The measured molecular weight of BS2 was 32048.979 Da. At the same time, the amino acid sequences (Table 1) of the five polypeptides of the BS2 protein were obtained by using on-line reversed-phase capillary column liquid chromatography electrospray tandem mass spectrometry CapLC-ESI-MS-MS, and Mascot was used to obtain meaningful searches in the secondary mass spectrum library, the results This protein was shown to have homology to two proteins (Bacillus amylolyticus neutral protease precursor HYBSN gi|67706 and Bacillus B16 neutral protease precursor bael6 AAV30844 gi|54126578)

[0037] These two homologous proteins both belong to bacillus neutral protease, and the homology between the two homologous proteins is as high as 89%. At the same time, the amino acid sequences of the fiv...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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 [0001] The invention relates to a bacillus subtilis B111 resistant to cotton wilt and verticillium wilt, which is derived from the BS2 protein of the bacterium and its related BS2 gene sequence. The bacterium and its BS2 protein have a certain inhibitory effect on the growth of cotton Verticillium dahliae and Fusarium wilt. The BS2 protein can be used in the research of biological pesticides or biological control agents. The BS2 gene can be used for research on the expression of disease resistance in transgenic cotton. Background technique [0002] Plant diseases are one of the main causes of world grain production reduction, causing 10% to 15% yield loss in crop production every year, and cotton loss is about 12% (Hainr and P.H.Schireier, Journal of.Pflanzenschutz-Nachrichten Bayer, (special issue) , 49(67):25-120, 1996). Wherein plant fungal disease is positioned at the first of plant four major diseases (fungus, bacterium, virus and nematode (Zheng Xiuf...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/31C07K14/32C12N15/63C12N15/82C12R1/125
Inventor 郭三堆白玮张锐
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap