Cloning,Functional Verification And Application In Production Of Fibroin Heavy Chain Gene In Sylepta Derogata Fabricius

Sylepta derogata Fabricius(Lepidoptera:Pyralidae),one of polyphagous insect pests,widely distributes in China.It can cause damages to a variety of economic crops.Silk fibroin heavy chain(FIBH)gene was selected to further study the spinning mechanism of S.derogata.Not only was the full-length cDNA sequence of FIBHgene of the S.derogata cloned,but also its expression at different developmental stages and fed on different hosts of the S.derogata was analyzed.Then the feeding method was adopted to interfere with the expression of FIBH gene in order to influence the leaf rolling behavior of S.derogata.The influences of interfering expression of FIBH on the development of S.derogata was also investigated.The main results are as follows:1.According to the known FIBH genes of other insects,the full-length cDNA of the FIBH gene of S.derogata was cloned by RACE.The full length cDNA of FIBH gene of S.derogata was 1465bp;the initiation codon was ATG,the termination codon was TAG and the opening reading fragment(ORF)was 1323bp.T According to the estimated amino acid sequence of FIBH gene,its molecular weight was 43.553 kDa and the isoelectric point pI was 6.26.Compared to that of other FIBH genes which had been registered in GenBank,the amino acids of the FIBH gene of S.derogata had 65%identity with those of Corcyra cephalonica,59%with Galleria mellonella and Antheraea assama.2.Using RT-PCR,the FIBH expression levels of S.derogata at different developmental stages and fed with different hosts was investigated.The results showed that the expression level of FIBH gene varied with the developmentalstages,and the expression level of FIBH gene in the second and fourth instar and prepupal stage was relatively higher compared to that in other developmental stages.The expression level of FIBH gene in the third and fifth instar larvae was lower than that in the previous instar larvae,and the expression of FIBH gene was very low,near to zero in the pupal stage and eclosion phase.The expression level of FIBH gene was different when S.derogata was fed with different hosts,and the relative expression of FIBH gene of S.derogata fed with piemarker was the highest,followed by cotton,hibiscus was lowest,and the differences between them was significant.3.Feeding method was used to interfere with the expression of FIBH gene.The experimental results showed that with the increase of the concentration of bacteria,the expression of the FIBH gene of S.derogata decreased.The FIBH gene expression decreased after the 2nd,3rd and 4th instar S.derogata larvae fed with bacteria,and the FIBH gene expression of S.derogata larvae fed with bacteria for 3 or 5days decreased significantly compared with that fed for only one day,the interference effect of the second and fifth instar was better than the third.All experimental results above showed that the concentration of bacteria,feeding time,and the larval instar can influence the interference efficiency of the FIBH gene.