The Cloning Of Peanut NBS Resistance Gene And The Impact Of Aspergillus Flavus Infection On Peanut

Peanut （Arachis hypogaea L.） is one of the four major oil crops in the world andalso the highest yield of oil crops in China with the total output accounting for45%ofthe world’s peanut production. Peanut is susceptible to infect by Aspergillus flavuswhich resulted in that there are excessive of aflatoxin in peanut, which had been alimiting factor of our peanut exports. Therefore, studies on resistant varieties ofpeanut should improve the understanding of resistance mechanisms to Aspergillusflavus and cultivating Aspergillus flavus resistance peanut varieties. NBS R genes arethe largest category of plant disease R genes. NBS conserved domain may becombined to the disease resistance signals’ transduction and played an important rolein plants disease resistance. Cloned NBS R gene from peanut may lay the foundationfor breeding of peanut Aspergillus flavus resistance varieties. In this study, we usingthe homology sequence cloning and RACE technology to clone NBS diseaseresistance gene from peanut and analysis it’s functions. It provided a foundation forcultivating Aspergillus flavus resistance peanut varieties. Meanwhile, we analyse theharmfulness of peanuts Aspergillus flavus infection by detecting aflatoxin content andpeanut quality changes after Aspergillus flavus infections.A NBS type R gene fragment PnAG₃was amplified with a pair of degenerateprimers which designed according to the NBS conserved regions of peanut. UsingRACE to isolate and clone the full length cDNA of gene PnAG₃. The result showedthat the full-length cDNA of PnAG₃was1876bp and contained a1335bp openreading frame. Multiple analyses showed that it had a certain homology with someresistance proteins, among which Arachis cardenasii resistance protein gene had thehighest homology （48.01%）. Sequence analysis showed the deduced PnAG₃proteincontains conserved NBS motifs such as P-loop, Kinase-2, Kinase-3a, GLPL andRNBS-C motif. Two pairs of real-time fluorescence quantitative PCR primers which according tothe sequence of PnAG₃gene and DF-actin gene respectively, were designed toconstruct the pMD18-T-PnAG₃and pMD18-T-Actin recombinant plasmid. And wesuccessfully established a SYBR Green I real-time fluorescence quantitative PCRmethod to precisely analysis the peanut PnAG₃gene expression level. Using thismethod we detected the PnAG₃gene expression changes in two different peanutvarieties with time changes. The result showed that the function of gene PnAG₃hascorrelation with the drought stress resistance and Aspergillus flavus resistance ofpeanut.Using real-time fluorescence quantitative PCR to analysis the relationshipbetween the function of gene PnAG₃and the peanut drought stress resistance which iscombining with the physiological index relative to drought resistance. The resultsshowed that the SOD, POD, CAT activity of anti-Aspergillus flavus species hasincreased3.15times,2.55times and4.95times in40⁵5%of field maximum watercapacity treatment group, respectively. But there is no significant difference in SOD,POD and CAT activity of susceptible species in the three treatment groups. MDAcontents are increased in all treat groups, and it is higher in susceptible peanut speciesthan anti-Aspergillus flavus species. Gene PnAG₃expression levels are higher inanti-Aspergillus flavus species than susceptible peanut species. Combined with thephysiological index relative to drought resistance and the PnAG₃gene expressionchanges, there is a correlation between the variation trend of gene PnAG₃expressionand adversity physiological indexes. And the gene PnAG₃expression in resistantvarieties was higher than susceptible varieties. So that, the function of gene PnAG₃isrelated to the peanut drought stress resistance.Using real-time fluorescence quantitative PCR to analysis the relationshipbetween the function of gene PnAG₃and Aspergillus flavus resistance which iscombining with the peanut quality changes. The results showed that after Aspergillusflavus infection, the expression changes of gene PnAG₃in J11has increased16.68times,11.16times and25.96times in seed coat, kernel and pericarp respectively. But it only increased2-3times in JH1012. This study is preliminary verified that thefunction of gene PnAG₃has the positive relationship with peanut resistance toAspergillus flavus infection. After Aspergillus flavus infecting, the peanut quality ofdifferent varieties has changed and the production has varying degrees of reduction.In addition, in this study we using Aspergillus flavus spore suspension infectpeanuts seeds to detect the aflatoxin content in peanut seeds and peanut qualitychanges with time changes. The result showed that the main part of Aspergillus flavusinfecting peanut is the seed coat. And the content of aflatoxin was increased with theinfection time increase. From largest to smallest of aflatoxin content in differentpeanut varieties was JH1012>HY22>J11. Meanwhile the contents of linoleic acid,oleic acid and fat were had a different degree changes.