Identification of Genes Related to Virulence of Fusarium Graminearum and Biopaning of DON Toxin Specific Affinity Peptides
|School||Nanjing Agricultural College|
|Course||Crop Genetics and Breeding|
|Keywords||Fusarium graminearum TAIL-PCR Deoxynivalenol Phage Display Peptide Library green fluorescent protein(GFP)|
Fusarium head blight or scab, caused by Fusarium graminearum, has been a very serious disease of wheat, barley and corn in China. Head blight not only causes the severe grain yield loss, but also causes the harvested grain contaminated with several mycotoxins. The consumption of food product made by grains contaminated with these toxins is a potential problem for human and farm animals.Mycotoxin Deoxynivalenol （DON） is a kind of trichohtecenes porduced by Fusarium. Although DON is one of the least acutely toxic trichothecenes, it should be treated as a serious food safety issue, beeuase it is a common contamination of wheat and corn, usually occurs in temperate climaets in years that are particluarly wet at the time of hvarest. DON causes feed refusal, emesis, immunosuppression or immunostimulation, terarogeny, cytotoxicity, reproductive toxicity and human cancers.The study is divided into two parts.To identify genes related to virulence, the virulences of 10 transposon insertion mutants of Fusarium graminearum were analyzed. Compared with the wild type,the virulence of mutant G12 had an obvious increase.It was also observed by GFP screening that mutant G12 spread faster in rachis than wild type. Flanking sequences of the transposon insertion site on the genome DNA was amplified by the TAIL-PCR technique.The blast result showed that the insertion position of transposon was located in a hypothetical gene （FGSG04134.3） in Supercontig 2,chromosomeⅡ. Protein structure analysis showed the protein contained C2H2 type zinc finger domain. 53% homology was found when compared protein sequence with Aspergillus fumigatus Af293 and Magnaporthe grisea C2H2 transcription factor con7. The function of FGSG 04134.3 was unkown. To screen for peptides that specifically bind to DON molecule from a phage-displayed peptide library. The DON antigen was used as the target molecule to screen the binding peptide from the Ph.D.-7 peptide library with phage display technique, and the positive clones were identified by ELISA. After four rounds of biopanning, the binding peptides were screened from the peptide library by ELISA and competitive inhibiting ELISA. Sequencing result showed that the binding peptides had high affinity and specificity. A peptide binding DON has been successfully obtained by screening the phage display library, which paves a way for improving ELISA detection of DON toxin.