Expression And Function Analysis Of Abcprotein Gene Of The Oriental Armyworm,Mythimna Separata (walker)

Mythimna separata(Walker)is a polyphagous insect,because of its habit of migration,the larger range of harm caused huge economic losses for China's grain production.With GM crops worldwide scale planting then reducing the use of chemical pesticides,while also control the relevant insect infestation efficiently.The vast majority of existing GM crops is transferred Bt(Bacillus thuringiensis)gene,because it produces insecticidal crystal proteins that have a specific insecticidal action against insects,but with the increase of pests selective pressure for pests,so its resistance problem has become increasingly serious.This study is selected the M.separata as the research object and has been obtain the full-length cDNA for M.separata ABC(ATP-binding cassette)transporter genes,determined relative expression of MsABCC2&MsABCC3 protein gene in M.separata in the different larvae and different tissues,also examine the function of ABC transporters by using RNA interference technology.The main results are as follows:1.The relative expression of armyworm larvaeMsABCC2 and MsABCC3 Gene?-actin as a reference gene,using the real-time quantitative PCR technology to examine the relative expression levels of MsABCC2&MsABCC3 from different larval instars and different tissues,and using the 2-??CT analysis method to analysis the data.The results are shown:the expression level of MsABCC2 protein gene was significantly different(P<0.05)in different instars of larvae.The relative expression of the 6th instar larvae is highest,but compared with the 4th or 5th instar larvae was not significantly different;the relative expression of the 3th instar larvae is lowest,but there was no significantly different for 2th instar larvae.For MsABCC3,the relative expression of 6th instar larvae is highest,and there were significant differences for the other instars of larvae,the relative expression of 5th instar larvae is lowest,and there was significantly different that compare with the 4th instar larvae,but there were no significantly different with the age of 2th or 3th instar larvae.Examine the expression levels of MsABCC2&MsABCC3 of 4?6 instar larvae in the midgut and in the rest of the larva body,then found the midgut significantly higher than in the rest of the larva body,then we know that MsABCC2&MsABCC3 protein gene was almost expressed in the midgut.2.MsABCC2 and MsABCC3 gene silencing and biological determinationAccording MsABCC2&MsABCC3 gene sequences were designed three siRNA,named si2-1,si2-2,si2-3 and si3-1,si3-2,si3-3.Using real-time quantitative PCR to detect the relative gene expression of MsABCC2&MsABCC3 after feeding on siRNA 24h,48h and 72h,the results showed that:after feeding si2-1 24h,the relative expression of MsABCC2 was significantly reduced that compared to the control;after feeding si3-1 or si3-3 72h,the relative expression of MsABCC3 gene significantly reduced that compared to the control.After we get the effective siRNA fragments then testing the M.separata sensitivity for Cry1Ac insecticidal protein after the genes have been silenced.The results showed that:the expression of MsABCC2 was suppressed after feeding on artificial diet containing Cry1Ac toxin,after 7 days,the mortality and weight of larvae was significantly lower than the control larvae that without MsABCC2 gene silencing.The results showed that the sensitivity to Cry1Ac toxin has related to the level of expression of MsABCC2,and the MsABCC2 protein of M.separata serves as a receptor of the Cryl Ac toxin.