Extracellular Protein Analysis Of Aflatoxin Synthesis Regulated By LaeA In Aspergillus Flavus

In order to study the aflatoxin biosynthesis regulatory mechanism by global regulator Lae A in Aspergillus flavus,the wild strain(AF)and lae A gene deletion mutant(no AF)of Aspergillus flavus were selected as the research object,the protein samples of two strains were extracted,SDS-PAGE technique and mass spectrometry were used to isolate and identify the different extracellular proteins,the target proteins associated with AF biosynthesis were confirmed,the mutant strains of Aspergillus flavus corresponding to the target protein were constructed.The main conclusions are as follows:(1)Eight kinds of extracellular differential proteins were isolated from the wild strain(AF)and lae A gene deletion mutant(no AF)of Aspergillus flavus.The fructose biphosphate aldolase precursor(fba A)and triose-phosphate isomerase precursor(tpi A)which were very important enzymes in the glycolytic pathway,xylanase precursor(xyn F3)and glucoamylase precursor(gla A)were mainly enzymes which related to the growth of Aspergillus flavus,the catalase B precursor(cat B)which was mainly related to the growth of Aspergillus flavus in the process of resisting the external oxidative stress,Amylase precursor,alkaline protease precursor,leucine aminopeptidase precursor,mainly related to nutrition intake,affected the growth of Aspergillus flavus.(2)RT-PCR technology was used to analyze the transcription levels of these eight differential extracellular proteins.The fructose biphosphate aldolase and triose-phosphate isomerase,xylanase and catalase of these four enzymes gene expression showed an increase trend in culture YES medium after 7 day cultivation;except for the expression of the glucoamylase gene began to decrease after 6 day cultivation.The expression of alkaline protease gene which was the highest in wild type strain(72 hour),showed a downward trend in Lae A deficient,the expression of amylase gene showed a rising trend in the wild type strain,while showed a downward trend in the Lae A deficient strains in wheat medium.The amylase,alkaline protease and leucine aminopeptidase of these three enzymes gene expression in wild type was the highest in wheat medium at 72 hour.(3)The vector of cat B and TPI gene knockout were successfully constructed.Then we knocked out the cat B gene of Aspergillus flavus CA14 and screened a mutant strain of cat B deletion successfully.Compared with the wild type of Aspergillus flavus,the mutant strain of cat B deletion has a strong tolerance and enhanced the capacity of toxin production through the analysis of TLC and HPLC.Its specific tolerance mechanism was still unknown.