Study On The Mechanism Of Antagonism Against Fusarium Gramineariumand Degradation By Bacillus Amyloliquefaciens MQ01

Wheat head blight widely spreads all over the world,which mostly produced by Fusarium graminearum species complex.It not only declines the production and quality of crop,but also produces many kinds of fungal toxin,such as deoxynivalenol and zeralenone,which are toxic to human and animals,decreases the body's immune function,and be carcinogenic,teratogenic and mutagenic.While traditional field experiment of resistant breeding is cumbersome and time-consuming,chemical agent has the risk of residues and resistance which pollute the environment,environment-friendly and biological prevention gradually becomes the potential method to control wheat head blight.The research object of this article is to study the antagonistic mechanism against Fusarium graminearium and zeralenone degradation of Bacillus amyloliquefaciens MQ01.The growth characteristics of Bacillus amyloliquefaciensMQ01 was studied.The growth conditions of strain MQ01 was optimized by single variable experiment.The result showed that the optimal growth temperature,pH,are 30?,6.0 respectively.MQ01 grew well when the volume of liquid is less than 100mL in 250 flask.The optimal carbon source and nitrogen source were glucose and peptone.In order to study the antagonistic mechanism against Fusarium graminearium and zeralenone degradation of Bacillus amyloliquefaciens MQ01,a mutant library of strain MQOlneeded to be constructed.As the strain MQ01 is a wild type Bacillus sp.,and pMarA which carring Tntransposon TnYLB-lis big,it's very difficult to transfer plasmid pMarA into the strain MQ01.The traditional Spizizen chemical method was first used,but it did not work.The electro-transformation method was then used to successfully transfer plasmid pMarA into the strain MQ01.In consideration of the low transferringefficiency,the transferring conditions were optimized.The optimal conditions were as follows:The cell concentration was OD600 = 0.9?1.0 and the transferring voltage is 2.0 KV.The optimized transferring efficiency was amount to 1.94?2.6×10-8.MQ01 was mutated by the transposon insertion mutation induced by high temperature and then a mutant library of strain MQ01,which was more than 5000 positive clones,was then constructed by inserting transposon TnYLB-1 of the carrier pMarA into the strain MQ01.The antagonistic mechanism of strain MQ01 against Fusarium graminearum was studied.Five positive mutants which lost antagonistic activity against Fusarium graminearum were screened by detecting antagonistic activity against Fusarium graminearum one by one.The flanking sequence adjacent to the transposon were cloned by Tail PCR method.By comparing the flanking sequence and the whole genome sequence,Five possible antagonistic related genes,such as:cytochrome p450,peptide 8,peptidase M4,subtilisin and chitin binding protein,were speculated.The five genes were then ligated to the expression vector pET-32a,and then transferred into E.coli BL21(DE3).The results showed that subtilisin gene was expressed successfully and got good antagonistic activity.In addition to being able to antagonize Fusarium graminearum,MQ01 also degrades the zearalenone(ZEN)produced by Fusarium graminearum.In order to have sufficient substrate ZEN to study the ZEN degradation of MQ01,the preparation,extraction process and detection technology of ZEN were studied.Fusarium graminearum 76F-25 was identified as the ZEN high-yield strain.Drying and milling method could extract ZEN with high efficiency.ZEN crystal with purity higher than 95%was obtained by using silica gel column,thin-layer chromatography,dextran gel column methods.HPLC and HPLC/MS methods detecting ZEN were established.MQ01 could degrade more than 90%of the ZEN within 7 days measured by HPLC.The ZEN degradation characteristics of MQ01 were as follows:strain MQOlcan degrade ZEN from 5 to 625?g/mL.The optimal temperature and pH for degrading ZEN were 30?,6.0?8.0 respectively.The aeration had no significant effect on ZEN degradation rate of MQ01 when the volume of liquid is between 25mL and 100 mL in 250 mL flask.But when the volume of liquid was more than 125mL,the ZEN degradation rate was decreased with the increase of liquid volume.Five mutants which lost the ZEN degrading ability were obtained by testing ZEN degradationability of the mutant library of strain MQ01.Further study needs to be done to clarify the ZEN degradation mechanism in the future.