Biocontrol Of Bacillus Subtilis Mrna Differential Display Analysis

Bacillus subtilis was a kind of important antifungal bacteria. They played an important role on controlling plant diseases. Bacillus subtilis strains Zh-2,Zh-4,Zh-6 and Zh-18, separated and conserved by Genetic Engineering Laboratory, Microbiological Institute of Heilongjiang Academy of Science, showed similarly morphological, physiological and biochemical characteristics and different antifungal activity against some plant pathogenic fungi. In order to discriminate these bacteria in molecular level and rapidly clone new gene, in this study we first utilized mRNA differential display technology to discriminate four Bacillus subtilis strains as well as looking for excellent gene.In this study, we extracted total RNA of Bacillus subtilis using TaKara RNAiso Reagent method, collected mRNA using MICROBExpress TM Bacterial mRNA Enrichment Kit. According to the rule that there is the similar GC content in E.coli and Bacillus subtils genomic DNA, 11 arbitrary primers were selected to amplify mRNA of different Bacillus subtilis. Results showed that 77 differential DNA fragments were obtained from mRNA of four bacterial strains. Five of them were sequenced and their functions were analysed using biological information method.S-1 DNA fragment, is from strain Zh-4, was 750bp in length. Its DNA sequence had 85% similarity with hypothetical protein related genes of B.subtilis strain 168.S-2 DNA fragment, is from strain Zh-4, was 462bp in length. Its DNA sequence had 99% similarity with 16S rRNA related genes of B.subtilis strain 168.S-3 DNA fragment, is from strain Zh-4, was 414bp in length.Its DNA sequence had 84% similarity with serine phosphatase related gene of B.subtilis strain 168.S-4 DNA fragment, is from strain Zh-18, was coincident with S-3 DNA fragment by BLASTx sequence alignment.S-5 DNA fragment, is from strain Zh-18, was 222bp in length.Its DNA sequence had 85% similarity with RNase R related genes of Bacillus pumillus SAFR-032.