Response Of Peanut Pericarp Genes To Aspergillus Flavus Infection And Expression Analysis Of AhRAF27 Gene

Peanut(Arachis hypogaea L.),which can be used for producing edible oil or eaten as food,is one of the main cash crops in China and the world.Aflatoxin contamination produced by Aspergillus flavus in peanut is the worldwide problem that seriously affects peanut production,trade and food safety.At present there are few studies on the molecular mechanism of peanut resistence to Aspergillus flavus at home and abroad,the mechanism of interaction between peanut and Aspergillus flavus,of peanut resistance to Aspergillus flavus and the number of genes related to the resistance is still unclear,which slow down the development of peanut varieties resistant to Aspergillus flavus.The aim of this study is to make it clear that how the Aspergillus flavus infection affected the expression profilie of the genes in peanut pericarp,clarify the expression characterization of AhRAF2 7 genes from NBS-LRR family genes related to Aspergillus flavus,and locate the protein product of AhRAF27 gene in cell.All these results will lay a foundation for the clarification of the molecular mechanism of peanut resistance to Aspergillus flavus,and genetic improvement of peanut resistance to Aspergillus flavus through genetic engineering method.The main results are as follows:1.Based on the high density of cDNA microarray with 101,344 unigene probes prepared before,the expression profile differences were analyzed after inoculated with 4spergillus flavus by comparing the hybridization results of gene chips using mRNA isolated from peanuts of Aspergillus flavus and drought treatment,peanut of drought treatment with the result of CK treatment.The results indicated that there were 4,917 genes showing more than three fold up-regulated expression and 5,055 genes showing more than three fold down-regulated expression.GO annotation and KEGG pathway analysis showed that the differences of the functions of differentially expressed genes in peanut pericarp were great,and the differentially expressed genes involved in metablic pathway were numerous.The important genes related to desease resistance,such as NBS-LRR family desease resistance genes,transcription factor genes and kinase genes,displayed differences in gene expression before and after peanut plants were infected by Aspergillus flavus.The differential expression of these genes might be probably in connection with the defense response of peanut after infected by Aspergillus flavus.hence they might be related to peanut resistance to Aspergillus flavus.From these genes,44 NBS-LRR genes were futher analyzed,the expression of 16 genes of them were found up-regulated,of 28 genes down-regulated,and among them there were 18 genes which showed at least five fold up-regulated or down-regulation expression.According to Blast2GO annotation results,these genes are resistance-related genes,therefore we speculated that they might be relevant to the infection of Aspergillus flavus.From these genes,11 genes which showed apparent induced expression by Aspergillus flavus were selected and verified by RT-PCR.The RT-PCR results were consistent with those obtained by gene chip analysis.The above results would provide gene resource for analyzing the molecular mechanism of peanut resistance to Aspergillus flavus.2.The NBS-LRR gene family is known as the biggest disease-resistance gene family which plays a key role in plant disease-resistance defense response.In this study,a down-regulated expression gene AhRAF27,which was induced by Aspergillus flavus,was obtained by gene chip analysis,the subcellular location analysis of the gene expression product protein as well as the analysis of phenotype of tobacco which transiently overexpressed the gene was carried out.The results showed that the expression product of the gene was seen in cytoplasm,and the transient overexpression tobacco did not exhibit significant hypersensitive response.However,the expression analysis of the important marker genes on the R pathway in the tobacco plants overexpressing the gene showed that the transient overexpression of this gene could cause the change of the expression of related genes on the R gene pathway.Therefore,it was suggested that this gene might be involved in the response of peanut resistance to Aspergillus flavus infection.