| Aspergillus flavus(A.flavus) can be found everywhere in the world, releasing its secondary metabolites-aflatoxins at the process of infecting grains, oil seeds, food and feed. Aflatoxin is proved to be a highly carcinogenic, teratogenic, and mutagenic fungi biotoxins. Peanut (Arachis hypogaea L) is an important oil crop worldwide but it is prone to be infected by Aflavus and contaminated by its products-aflatoxin. Continuous high temperature and drought pre-harvest of peanut, as well as hot and humid at post-harvest, making the aflatoxin contamination even worse. Aflatoxin contamination of peanut is dangerous to the health of human and animal, it has been one of the most important barrier to the industrial development of peanut, so prevention of aflatoxin contamination has important economic and social significance. The application of resistant varieties was considered to be an efficient way to solve aflatoxin contamination of peanut. Due to the scarcity of germplasm resources with high efficiency and stable resistance to the infection of A.flavus, hindering the breeding of peanut for disease-resistant. Therefore, making intensive studies of the mechanisms that peanut resist Aspergillus flavus invasion and isolating disease resistance associated genes, is an effective measure for disease resistance breeding and then to control aflatoxin contamination.For plants, NBS-LRR disease resistance genes play central roles in recognizing pathogens and initating defense reaction, these genes have broad application prospects in the molecular breeding of plants resistant to disease,insect pests and various stress. However, NBS-LRR class R genes related to the resistance against A.flavus in peanut has not been reported. Because the genome of peanut is complex, it’s difficult to isolate genes conferring the resistance to A.flavus from peanut. So, screening out NBS-LRR genes in peanuts that were induced by A.flavus through peanut high-density microarray is a simple but effective way. Based on the analysis of peanut microarray, we performed a series of researches, the results were the followings:1. Based on the analysis of peanut microarray, we screened out a NBS-LRR class gene, which was up-regulated by A.flavus under drought stress. Using RACE method, we cloned the full-length sequence of this gene, designated:AhRAF4. It was 3484bp in length including a 2808bp ORF, and 3’,5’ untranslated regions; there was no intron in its gDNA. Structural and functional analysis of the protein encoded by this gene showed, there were NB-ARC, LRR and P-loop conserved domains in AhRAF4, and they shared high similarity with NBS-LRR class resistance protein in the soybean, alfalfa, castor and other crops. Semi-quantitative RT-PCR showed this gene mainly expressed in roots,leaves and peel, and it was apparently up-regulated by A.flavus. In addition,the results of real-time PCR showed:AhRAF4 could be induced by MeJA, which was a Signaling molecule could stimulate the expression of plant defense gene. The differential expression of AhRAF4 gene in four groups(infected A.flavus with drought, drought stress alone, infected Aflavus under normal condition, untreated control) of resistant genotype Xinhui and susceptible genotype Yueyou 92 were different in the change trends. And at the primary stage of inoculation, the synergistic induction of A.flavus and drought stress to AhRAF4 gene in the susceptible genotype was more apparent than that in the resistant genotype. At the late stage of inoculation, the change trend of differential expression turned gentle. So, we speculated that AhRAF4 gene might involve in the recognition the infection of A.flavus. Under normal irrigation conditions, no matter resistant genotype or susceptible genotype, there was no significant differences in gene expression, we speculated that drought stress might promote the recognition function of AhRAF4 gene.2. In order to get more comprehensive knowledge of the molecular mechanisms of peanut resisting A.flavus, we also screened out a NBS-LRR class gene-AhRAF42, which was down-regulated by A.flavus under drought stress. We got six copies in different length of AhRAF42 by RACE, structural analysis showed all of them shared functional domains with other R proteins, such as NB-ARC, LRR, P-loop and so on, and they had almost same physical-chemical characteristics. AhRAF42 was homologous to Rpsl-k clusters belonged to NB-LRR type gene and conferred resistance to Phytophthora sojae, we speculated AhRAF42 gene might be a disease-resistance gene family. Expression analysis of AhRAF42 gene by semi-quantitative RT-PCR showed AhRAF42 mainly expressed in root, peel, testa where were prone to be infected by A.flavus, and it was apparently down-regulated by the infection of A.flavus with drought stress. We assessed signaling requirements for the function of AhRAF42, it was slightly down-regulated by exogenously MeJA and it was free from the effects of other signaling moleculars. Real-time PCR was performed to analyze the differential expression of AhRAF42 gene in resistant and susceptible genotypes. The change trends of differential expression between treated group(inoculated A.flavus under drought stress,inoculated A.flavus under normal condition)and untreated group(drought control,untreated control) were different in resistant and susceptible genotypes. Under drought stress, the down-regulation of AhRAF42 gene in the resistant genotype was more apparent than that in the susceptible genotype; without drought stress, the up-regulation of AhRAF42 gene in the susceptible genotype was more apparent than that in the resistant genotype. So we speculated AhRAF42 gene was involved in recognition and response to A.flauvs, but drought stress may hinder these reaction. In addition, subcellar location displayed: AhRAF42 gene mainly located in the membrane, this might be associated with the function of recognition. |
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