| Long-term heavy use of chemical pesticides destroyed the environment and ecological balance,was also a threat to food safety,while biological pesticides is security for human and animal,has no pollution to e nvironment,is conducive to the sustainable development of agriculture,is the inevitable trend of pesticides industry.Bacillus has advantages of strong resistance and convenient for making into various dosage forms,is an important source of biological pesticides.Bacillus atrophaeus was mainly used in detecting disinfectant and controlling the quality of sterilization,but there are few reports in the prevention of plant diseases.The strain NMB28 that was isolated from the soil samples of Inner Mongolia was identified as Bacillus atrophaeus,by observing the morphological characteristics and physiological and biochemical characteristics,and combining with the analysis of 16 S r DNA,gyr A,rpo B and ITS sequences;Using tablet standoff method and double culture method to determine the bacteriostatic spectrum,the strain NMB28 inhibited a variety of plant pathogenic fungi and bacteria;The results of control effect measurement in vitro show that the fermentation liquor strain NMB28 can effectively prevent and control Botrytis cinerea;Through the transparent circle method detected,strain NMB28 can produce siderophore,chitinase and glucanase,protease and amylase;The strain NMB28 can form strong biofilm and produce IAA;The chitinases of strain NMB28 were inducible enzymes,shaking culture for 120 h enzyme activity reached the highest,and chitinases can effectively inhibition Botrytis cinerea spores germination.The crude proteins of fermentation broth of strain NMB28 by ammonium sulfate precipitation,were detected by SDS-PAGE that strain NMB28 produced two kinds of chitinases by chitin powder induced.Based on the reported chitinase genes of Bacillus atrophaeus,primers were designed to amplify the chitinase genes chit1 and chit2 from the strain NMB28.The analysis of gene sequence s and amino acids coding protein showed that the sizes of chit1 and chit2 were respectively 1791 bp and 2091 bp.C hit1 and Chit2 belonged to GH18-chitinase-like superfamiliy,had the signal peptide,and existed the typical domain of Glyco-18 family members in common.chit1 and chit2 respectively connected with expression vector p ET-30a(+)and successed in prokaryotic expression.The size of C hit1 was about 65 KDa in line with expectation,but natural chitinase was about 50 KDa,so Chit1 may be cut in strain NMB28.C hit2 was expressed inclusion body of about 80 KDa consistent with expectation.The possible reason is that the expression vector was not appropriate.C hit1 had little effect on inhibiting spores germination of Botrytis cinerea.Because C hit2 was not soluble expression,the antimicrobial activity of C hit2 was not determined,so C hit1 may play a supporting role or inducing factor.The optimum temperature for Chit1 was 60 ?.C hit1 was sensitive to high temperature.Fe2+,Fe3+,Cu2+,Co2+,Mn2+,Ca2+ seriously inhibited the enzyme activity of C hit1,Na+,Mg2+,EDTA could improve the activity of enzyme.The glucanase gene b Glu from strain NMB28 was 732 bp,encoding Glyco-hydrolase-16 superfamily.b Glu contained the signal peptide region and glucanase catalytic region of Glyco-hydrolase-16 superfamily C-terminal.Recombinant protein b Glu with the size of about 28 k Da was obtained by prokaryotic expression in E.coli BL21(DE3).b Glu had a higher activity,but antifungal activity weak.Strain NMB28 was sensitive to most antibiotics,then the transformants,that plasmid p GFP-4412 was transformed into strain NMB28,were selected with kanamycin.The positive transformants were identified by colony PCR,emited green fluorescence under fluorescence microscope.The success of GFP marked strain NMB28 provided the basis for the study of colonization on plants. |
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