Detection Of Antagonism-related Genes Of Bacillus Subtilis EDR4Using A TnYLB-1Transposition Insertion Library

With the development of food safety and environmental pollution problems, manycountries have already strictly limited some of chemical fungicides, so biological control ofplant diseases has been more and more concerned around the world. Using geneticengineering methods to reconstruction biocontrol bacterium can be improve the ability toproduce antagonistic substances and biocontrol effect. Bacillus subtilis EDR4, isolated fromwheat roots by our laboratory, has high control effect on some pathogenic fungus such asGaeumannomyces gramini and Sclerotinia sclerotiorum. In order to make EDR4become apreparations commodified for large-scale use, we should optimiza or transform EDR4. In thisstudy, transposon TnYLB-1transposon mutagenesis was used to a further study on Bacillussubtilis EDR4. The main results were as follows:1.The plasmid pMarA which carries transposon TnYLB-1was transformed into Bacillussubtilis EDR4by using chemical transformation. According to plasmid amounts,co-cultivation times and a orthogonal experimental containing three culture times, atransformation system for EDR4was constructed, the transformation system was that: theOD600of seeding solution was1.2to1.5, the OD600of bacterial in culture medium one was1.3to1.5, the time of culture medium two was2hours, plasmid amounts was500ng,co-cultivation times was90min.2. The insertion mutant library containing2756mutants was constructed under thecondition of50℃, PCR amplification analyses of14mutants randomly selected from themutant library of Bacillus subtilis EDR4showed that the transposon TnYLB-1had insertedinto EDR4genome. The result of southern hybridization showed that the transposonTnYLB-1had inserted into Bacillus subtilis EDR4randomly, the mutants were inserted by asingle copy,and also some multiple copies appeared.3.Two mutants, EDR4-T1272and EDR4-T1473which the activity of againstingSclerotinia sclerotiorum significantly decreased, were screened from640mutants, thetransposon did not affect the biological characteristics of EDR4and can be stably inherited inthe mutants.4.The transposon insertion flanking sequences of EDR4-T1272and EDR4-T1473were cloned by IPCR amplification. Homology alignment revealed that the flanking sequences ofEDR4-T1272showed high homology to the glycosylase gene yvbX of BS168genome and theflanking sequences of EDR4-T1473showed high homology to a hypothetical protein-codinggenes of BS168genome, these two genes may be associated with the antibacterial activity ofEDR4.