Cloning and expression of Plutella xylostella(Linnaeus) cuticular protein gene

ObjectivePlutella xylostella(Linnaeus)cuticular protein gene(PxyICP)was cloned,its sequence cha-racters and developmental expression pattern were analyzed to provides reference for the physiological function of PxyICP in the formation of cuticular in P.xylostella.MethodP.xylostella was used as materials,PxyICP was cloned by reverse transcription polymerase chain reaction(RT-PCR)and rapid amplification cDNA ends(RACE).PxyICP sequence charac-ters were analyzed by bioinformatics method.The relative expression levels of PxyICP mRNA were investigated by real-time fluorescent quantitative RT-PCR method at different develomental stages.ResultThe cloned PxyICP contained 531 bp open reading frame in length,encoded 177 amino acids. Its relative molecular mass was 18.90 kDa,and isoelectric point was 5.32.Amino acid sequence analysis showed that the 1stto the 16thamino acids of cuticular protein was a signal peptide that participated in transmembrane protein transfer.This sequence presented typical characteristics of RR-1 conser-vative base sequence in the CPR family of insect cuticular protein.According to real-time fluorescent quantitative PCR analysis at various development stages,expression of PxyICP differed at each developmental stage.In 2ndinstar larva,3rd instar larva,4thinstar larva,pupal and adult,expression levels of PxyICP were 2.43,0.51,0.63,3.19 and 12.32 times as that in 1stinstar larva.And the expression level reached the peak in adult.ConclusionGene PxyICP is cloned successfully. According to its development expression pattern,protein PxyICP is involved in the cuticle sclerotization and melanogene-sis for P.xylostella adult.