Transcriptional Analysis Of Gene Clusters Involved In The MEP Pathway In Bacillus Subtilis And Construction Of The Recombinant Strain

Bacillus subtilis is the perfect biocontrol bacteria,used in the farming research.In Bacillus subtilis,although researchers have elucidated many of the structures and functions of proteins encoded by genes involved in the MEP pathway,few studies have reported the transcriptional profile of genes involved in the MEP pathway,and few studies have reported the construction of engineering strain with the character of effect the behavior of natural enemies of insts.Accordingly,in this study,we aimed to elucidate the presence of MEP pathway genes in the genome of B.subtilis and construct the strain with gene to affect the behavior of natural enemies of insts.Bioinformatics results showed that MEP pathway genes and gene coding FPP synthesis enzyme were distributed throughout the genome of the plant-associated B.subtilis 916 strain.Amino acid sequence Similarity was analyzed by BLAST,and the result identify the amino acid sequences of the nine genes(dxs,dxr,isp D,isp F,ip K,isp G,isp H,idi,and isp A)up to 73%.The growth curve of Bs916 in LB medium was detected,and the result showed these genes were transcribed at both the exponential and stationary phases through RT-PCR method.We further confirmed the transcription units of dxs,dxr,isp D,isp F,ip K,isp G,isp H,idi,and isp A genes in B.subtilis 916 through reverse transcription polymerase chain reaction analyses.The results showed that dxs was cotranscribed with isp A and other five genes becoming an operon with size of 6352bp;dxr was transcribedand with other 19 genes becoming an operon with size of 19355bp;there was only 8bp between isp D and isp F,and were cotranscribed with other 20 genes becoming one operon with size of 18580bp;idi was transcribed with other one gene becoming one operon with size of 3413bp;ip K was transcribed with other 4 genes becoming one operon with size of 2535bp;isp G was transcribed with 2 genes locating the upstream of isp G becoming one operon with size of 2954bp;and isp H was transcribed with one gene locating the downstream of isp H becoming one operon with size of 819 bp.Meanwhile,the transcript start site of the dxs operon was determined by 5?RACE method,which is “C” nucleotide residue located in the upstream of gene named KO64_RS11240 in Gen Bank,and 157 bp from translation initiation codon ATG.The strain with the ability to affect the behavior of natural enemies of insts was constructed preliminarily by genetic manipulation.The results showed that the optimized gene PITPS4 could be transcribed successfully in B.subtilis 916.Our study provided a molecular basis for investigating the synthesis of homoterpenes in the MEP pathway in B.subtilis 916.Meanwhile,our study provided a basis for constructing the efficient engineering strain with the ability of effect on the behavior of natural enemies of insects.