Screening And Identification Of Antagonistic Bacteria Of Cotton Rhizosphere, Cloning Of Iturin A Operon From Bacillus Subtilis MH25

Thirteen samples of cotton rhizosphere were gathered from fields in Jinxiang and Jining Academics of Agricultural science. Bacterial isolates of 1277 were obtained by plate culture . Twenty five isolates antagonistic against Rhizoctonia solani Kuhn were screened by hyphal extension inhibition assay. Isolates MH1 and MH25 have obvious antibiosis and the potential as biocontrol agents. According to the morphological characteristics, physiological and biochemical properties and phylogenetic analysis of 16S rDNA, we identified the strain MH1 and MH25 as Brevibacillus brevis and Bacillus subtilis, respectively.Based on the conserved sequences of NRPSs of knowned antibiotic operons, we designed primers TGD(5'-TWYCGIACIGGIGAYYKIGKICG-3') and LGG (5'-AWIGARKSICCICCISSRIMRAARAA-3') and obtained 818bp DNA fregment from Bacillus subtilis MH25. We analysised the high homology operons iturinA, bacillomycin and mycosubtin. Twenty pairs of primers were designed to clone the related operon from Bacillus subtilis MH25. Finally, we obtained a 38845bp DNA sequence. Multiple-alignment analysis and phylogenetic analysis were performed using DNAman software. It was found that the 38845bp DNA sequence has four open reading frames(ORFs). Homology of them to ituD, ituA, ituB and ituC of were 99%, 98.7%, 98.99% and 99.48%, respectively. we predicted a promoter region upstream of ORF1, which contains TATACACA-16bp-TAGGAT sequence. The characteristic sequene is different from the consensus -10 and -35 (TTGACA-17bp-TATAAAT) sequences ofσA.In order to identify function of the tryptophane adenylation domain in ituB of Bacillus subtilis MH25, the experiment of pop in-pop out was carried out as follows. Firstly, about 800bp DNA fragment containing tryptophane adenylation domain were amplified from Bacillus subtilis MH25 by PCR, and then inserted into cloning vector pMD18-T to construct recombinational plasmid pMDT. Secondly, plasmids pMDT and pBRMCS-5 were cut with both SalⅠand XbaⅠ, respectively, and then the aimed DNA fragment was inserted into pBRMCS-5 to obtain the recombinational plasmid pBRMT. Thirdly, one about 1.2kb PstⅠfragment congtaining Km gene from pUC4K was inserted at the PstⅠsite of pBRMT to construct the recombinational plasmid pBRMTK. Lastly, we amplied the tyr::Km fragment from pBRMTK and transformed it into the protoplast of Bacillus subtilis MH25, then plated them onto CMR agar containing 30μg/mL Kanamycin. Unfortunately, no transformant has been obtained.