Identification And Characterization Of Novel Antimicrobial Protein APn5 From Bacillus Subtilis Strain BSn5

The research includs two parts:one is identification and characterization the novel antimicrobial protein APn5 isolated from Bacillus subtilis strain BSn5;another is to construct the restriction maps of plasmids pBMB69 and pBMB95 in Bacillus thuringiensis strain YBT-1520.1.Bacillus subtilis strain BSn5 was isolated from Amorphophallus konjac calli contaminated by bacteria.BSn5 could inhibit Erwinia carotovora subsp.carotovora strain SCG1,which causes Amorphophallus soft rot disease and affects Amorphophallus industry development.The extracellular proteins was isolated from the BSn5 culture by ammonium sulfate precipitation with 30%relative saturation and further purified by ultrafiltration.There was only a 31.6 kDa band could be detected by SDS-PAGE,which was named as APn5.APn5 showed a narrow inhibition spectrum,showing low inhibition activity to plant pathogenic fungus and strong inhibition activity only to Erwinia carotovora subsp.carotovora and Rhodococcus fascians.APn5 was sensitive to high temperature and alkaline pH,pronase E,and partial sensitive to trypsin and proteinase K. During strain BSn5 growth phase,the inhibition activity was unstable,which would gradually lose on stationary phase and disappeared finally.After investigating all Bacillus subtilis strains stored in our lab,only B47 produced a protein with the similar molecular weight and inhibition activity to APn5,and the protein was named as APn5-like.APn5-like showed positive signal during the Western blot using the antibody prepared by APn5.MALDI-TOF-MS and Q-TOF analysis showed that there was no high relative to known proteins from Matrix Science database.There were two pairs of identical peptides in APn5 and APn5-like,all of that had similarity to N-terminal sequence of Bacillus subtilis flagellin;in addition,two peptides in the APn5 and APn5-like show functional similarity,respectively being similar to DnaJ and metal-binding protein.On the basis of the difference on inhibition spectrum and characteristics,APn5 and APn5-like are allied to a novel antimicrobial protein. 2.Bacillus thuringiensis strain YBT-1520,which is highly toxic to lepidopteran pests, was isolated from the soil and protected by patent.The whole genome of YBT-1520 was sequenced by shot-gun sequencing,and the preliminary analysis of the sequence revealed that there were 10 plasmids harbored in YBT-1520.Because of the insertion sequence and repeat sequence,there was discrepancy between the different sequence splice results in whether pBMB69 and pBMB95 were two different plasmids or they should be joined together as one plasmid.This work tried to clarify this discrepancy by constructing the restriction maps of plasmids pBMB69 and pBMB95.The BAC library of YBT-1520 total DNA was screened by PCR using the primers designed on the basis of ori44,ori60 and cry1Ac10 sequence. Finally,there were 9 clones containing ori44 and cry1Ac10,and only 1 clones harboring ori60 and cry1Ac10.The clones EMB2H05(contain pBMB69 fragment) and EMB2D10 (contain pBMB95 fragment) with the largest inserts were selected and digested with the appropriate restriction enzymes.We estimated the size of the restriction fragments separated by pulsed field gel electrophoresis and agarose gel electrophoresis,and at last, constructed the restriction maps of the whole pBMB69 and the repeat region of pBMB95. Compared with the sequence splice results,there was a 40 kb reversal fragment in pBMB69;the repeat region in pBMB95 was in accordance with the sequence splice results.According to the restriction maps,pBMB69 and pBMB95 were two independent plasmids.