Cloning,Expression And Efunctional Verification Of Fibroin P25 Chain Gene In Sylepta Derogata Fabricius

Sylepta derogata Fabricius,Lepidoptera:Pyralidae,is one of polyphagous distributed insect pests.In order to expoud the silk spinning mechanism,the silk fibroin P25 gene was studied in this paper.The whole length of the cDNA sequence was obtained by gene cloning,then the expression levels of the fibroin P25 gene in different development stages and different hosts were detected and analyzed.On this basis,the feeding method was used to interfere with the expression of silk fibroin P25 gene by RNAi in order to carry out functional verification.The main results were as follows:By comparing the fibroin P25 gene of other known lepidopteran insects,the full length of P25 gene of S.derogata was cloned by RACE.The whole length of cDNA P25 gene is 859bp.The starting codon is ATG and the terminating codon is TAG.The whole length of the coding region is 663bp.The whole length of the gene was submitted to GenBank with the login number KY792994.The nucleotide sequence of the fibroin P25 gene was converted to its amino acid sequence and analyzed.It was estimated that the molecular weight of the protein was 25.653kDa and the isoelectric point pI was 8.02.The whole length of the gene was compared with other insects fibroin P25 genes which had been logged in to GenBank.The result showed that the 63%identity was the highest with Galleria mellonella,followed by Corcyra cephalonica,and the identity was 62%.Real-time quantitative PCR technique was used to test the Expression level of fibroin P25 gene in different development stages and different hosts.The results showed that the relative expression of fibroin P25 gene was closely related to the insect ages and worm status.The relative expression of P25 gene had a peak in the 2L larvae and pre pupae stages and the pre pupal stage was higher.The expression of the 1st、3rd、5th instar larvae’s fibroin P25 genes were lower while the expression of the P25 gene rapidly decreased in the pupal period and after the emergence of the silk fibroin P25.The large roll of cotton on the leaf of the vane is fed.The relative expression level of fibroin P25 gene in the 2 instar larvae of the leaf borer was the highest,followed by that of the two species.The expression of P25 gene dsRNA of cotton leaf roller was interfered with the expression of larval target genes by feeding method.The results of different concentrations of bacterial solution showed that the relative expression level of dsRNA bacteria decreased.After feeding we found that the the expression were decreased especially in 5th larvae while the 3rd was higher than others.The results of the experiment on different hosts fed with bacterial liquid showed that the expression of silk fibroin P25 gene in the 2ed instar larvae of cotton roll stem borer on different hosts decreased,and the expression of silk fibroin P25 gene was significantly decreased.The above results showed that the concentration of bacteria solution feeding time,feeding age and the feeding host all could affect the interference efficiency of fibroin P25 gene.