Screening Of Deoxynivalenol Toxigenic Strains And The Possible Toxigenic Molecular Mechanism

Deoxynivalenol（DON）, also known as vomitoxin, is the secondary metabolite mainly produced in the growth process of Fusarium graminearum, Fusarium culmorum and other Fusarium spp. As one of the worldwide contaminants, DON has cell toxicity, immune toxicity and genetic toxicity typically. In china, DON can be detected in wheat, corn and other cereals each year with a concentration exceeding the current national standards, which is seriously affecting the quality and the safety of agricultural products.Given its universal presence in grains, it is of great significance to make researches on the pathogenic mechanism of Fusarium spp, the biosynthesis pathway and the possible toxigenic molecular mechanism of DON. By regulating the metabolism of pathogenic strains specially, the synthesis of DON can be reduced.Taking high-toxigenic F.graminearum Fg1 and non-toxigenic F.graminearum Fg2 as the research objects, this paper has studied the biosynthetic pathway and the molecular mechanism in the production process of DON from F.graminearum. The main research results are as follows:1. By combining the specific primers screening method with HPLC method, seven strains of F.graminearum were screened from wheat scab grains, including six DON-producing strains and one DON-negative strain. The 5.8S rDNA-ITS sequences were sequenced between DON high-toxigenic strain Fg1 and DON-negative strain Fg2 and these two strains were identified as Fusarium graminearum.2. Different factors influencing the growth and the toxigenic amount of F.graminearum Fg1 were analyzed under laboratory conditions, including such factors as different carbon sources, nitrogen sources, inorganic salts, temperature, pH, etc. The results showed the growth and the toxigenic amount of F.graminearum Fg1 differs greatly in different culture conditions. The strains grew best and had the highest toxigenic amount of DON in the culture condition of dextrin as the carbon source and yeast extract as the nitrogen source at pH 8.0and 25 ℃.3. The differentially expression proteins were compared between F.graminearum Fg1 and F.graminearum Fg2 by using proteomics. The comparison showed that there are great differences between the two strains in such proteins as proteins related to basic metabolism,oxidation reduction equilibrium proteins, secondary metabolism proteins, signal transduction proteins and regulatory proteins. There was an obvious increase of glyceraldehyde-3-phosphate dehydrogenase（GAPDH）, 2,3-phosphoglycerate mutase（PGAM）, pyruvate dehydrogenase（PDH）, flavin adenine dinucleotide monooxygenase（FADMO）, terpenoid synthase（TPS） and 4-diphosphocytidyl-2-C-methyl-D-erythritolkinase（CMK） in F.graminearum Fg1 expression.4. The differentially expression genes of F.graminearum Fg1 and F.graminearum Fg2 were compared by using transcription analysis technique and the twelve genes related to DON biosynthesis were tested by using qRT-PCR. The results showed that eleven genes were positive and one negative in regulating DON biosynthesis. In F.graminearum Fg1 expression,there was an obvious increase in terms of encoding glycolytic pathway, terpenoid biosynthesis pathway and MAPK metabolic pathway. These genes include coding glucokinase gene（FGSG₀8774）, 2,3-phosphoglycerate mutase gene（FGSG₀6055）,phosphaglycerate kinase gene（FGSG₀3992） and enolase gene（FGSG₀1346） in encoding glycolytic pathway, O-acetyltransferase gene（FGSG₀3124）, coding acetyl-coenzyme A synthetase gene（FGSG₀0330）, Isotrichodermin C-15 hydroxylase gene（FGSG₀3191）,cytochrome P450 monooxygenase gene（FGSG₀1284） and bifunctional solanapyrone synthase gene（FGSG₁2082） in terpenoid biosynthesis pathway and coding serine threonine protein kinase Psk1 gene（FGSG₀0469） and serine threonine protein kinase Cek1 gene（FGSG₀1312） in MAPK metabolic pathway. There was an obvious decrease of transcription factor Atf1 gene（FGSG₁0142） expressed in F.graminearum Fg1.