Isolation And Expression Of Peanut Anti-Aspergillus Flavus Related Gene

Peanut is an important economic and oil crop in China, and it is one of the most vulnerable crops to Aspergillus flavus infection, the infection caused by Aspergillus flavus pollution not only does directly harm to people's health but also effects on quality of peanut and foreign trade export. So people always try to take various measures to prevent Aspergillus flavus pollution effectively.The application of resistance varieties is the most direct and effective method to control disease, because the scarcity of germplasm resources of anti-Aspergillus flavus peanut together with the long cycle of conventional breeding makes resistance varieties far from satisfaction to the needs of production. With the rapid development of biological technology, exogenous resistance gene is introduced into peanut by using gene project, which brings new hope for resistant breeding.In this study, different expression genes were separated from two cultivars of resistance and susceptibility by Gene chip technology combined with real-time PCR technology, obtained full-length gene by using RACE technology, and constructed the prokaryotic expression vector, which laid the foundation for cultivate new varieties of peanut resistance to Aspergillus flavus. The main results of research as follows:1.The existing 61078 group of soybean hybridization probes were used to hybridise resistant and susceptible varieties of peanut. The results show that the seed coat of J11 relative to jinhualO12 has 152 genes which were up-regulated expression and SLR>1, and 236 genes which were down-regulated expression and SLR>1. In the research, 75 genes were isolated, the function of some genes are known, others are similar to, through the anlysis of different genes expression using BLAST.2.Using modified Trizol One-step method, extracted RNA from the seed coat of resistant varieties J11 which were infected by Aspergillus flavus for 10d, 20d, 30d (started to infect and set up the control group 40days before harvest). Based on the results of gene chip, 13 genes selected were detected by using real-time PCR, results showed that there were 6 different expression genes before and after infected. In this paper, we researched these genes.3.The AW86 full-length gene sequence is got by using RACE technology from extracted high-quality RNA, it contains 828bp and a 666bp ORF. It is found that after analysis Amino Acids encoded by the gene ORF has a conserved sequence of Catalase in plant and high homology with soybeans, chickpeas and other legumes. Transcription factor containing catalase domain is related to signal transduction of physiological and biochemical reactions. It joins in the gene expression of insect and disease resistence and obtained protein, so the protein could be used to improve the resistence to Aspergillus flavus of peanut.4. According to obtained full-length sequence , we design primers with restriction sites, amplify ORF fragment of AW86 gene with PCR technology and make it connect with expression vector PGEX-T4-1, introduced into E.Coli BL21 and induced expression, then through SDS-PAGE gel electrophoresis analysis of protein products, obtained a specific expression product which molecular weight is 70KD, it shows that full-length target genes contain a complete cds. It still needs further study.