Molecular Characteristics And Functional Analysis Of CYP4 And CYP303A1 Genes From Locusta Migratoria

The cytochromes P450, a diverse class of enzymes found in virtually all insect tissues, are involved in many important biological processes including the synthesis and degradation of ecdysteroids and juvenile hormones, and even the metabolism of xenobiotic compounds. In this study, we utilized Locusta migratoria as a model and cloned 3 CYP4 genes, CYP303A1 and a cytochrome P450 reductase gene. Moreover, the molecular characteristics and biological functions of those genes were analized for providing insight into pest control.1. Molecular characteristics and biological functions of 3 CYP4 genesThe full-length sequences of three novel genes named CYP4C69、 CYP4C73 and CYP4DH1 were successfully cloned by RT-PCR from the locust (GenBank accession numbers:KF857162, KF857163, KF857164). Furthermore the mRNA expression levels of CYP4C69, CYP4C73 and CYP4DH1 were analyzed in different developmental stages and tissues by real-time quantitative PCR. The results showed that the expression level of CYP4C69 was higher in fifth instar nymph than in the adult stage. Moreover, the expression levels of CYP4C73 and CYP4DH1 during the whole developmental stages were relatively higher in first, fourth and fifth instar nymphs and in adult stage. The results of CYP4 genes expression from different tissues indicated that all of the 3 genes had a high expression level in gastric caeca, CYP4C69 and CYP4DH1 highly express in fat body. As CYP4C69, CYP4C73 and CYP4DH1 transcripts were significantly repressed in locusts by RNAi, we assessed the susceptibility of the dsRNA injected locusts to different insecticides, such as malathion, chlorpyrifos and deltamethrin. It was found that CYP4C69, CYP4C73 and CYP4DH1 expressions were significantly silenced in locusts after dsRNA injection. The susceptibility of locusts to malathlon, chlorpyrlfos and deltamethrin suggested that there was no marked difference between locusts injection of dsCYP4C69, dsCYP4C73 and dsCYP4DH1 and dsGFP.2. Analysis of CYP303A1 FunctionsThe cDNA sequence of CYP303A1 was searched by bioinformatics method, and confirmed by RT-PCR. A phylogenetic tree was constructed using the homologous sequences of CYP303A1. mRNA expression patterns of CYP303A1 in different tissues and developmental stages in L. migratoria were performed by real-time PCR, then the RNA interference (RNAi) was conducted to study biological function of CYP303A1 in L. migratoria.The full length cDNA sequence of CYP303A1 contains an open reading frame of 1488 nucleotides encoding 496 amino acid residues. The results of tissue-specific expression indicated that CYP303A1 was expressed in all of the major tissues and the expression level in foregut and integument were higher than in midgut, gastric caeca, hindgut, malpighian tubule and fat body in 5th instar nymphs. In addition, CYP303A1 was less expressed during the first 6 days after molting with the highest expression in the integument of fifth instar nymphs at the seventh day. The results of RNAi showed that the expression of CYP303A1 was significantly reduced for 70.5% after injecting dsCYP303A1 for 24 hours. Compared with controls injected dsGFP, the insects injected with dsCYP303A1 was unable to molt the old cuticle, and finally died. In contrast to the control groups, the mortality of fifth instar nymphs injected with dsCYP303A1 was up to 92.5%.HE staining of integument in the fifth instar developmental stage showed that the new cuticle of locusts treated with dsCYP303A1 was thinner than the controls. Additionally, real-time PCR was used to test the expression levels of key genes involved in chitin biosynthesis pathways, such as Gfat, UAP, CHS and GNA. The results indicated that the expression of Gfat in locusts treated with dsCYP303A1 was significantly decreased than the controls. Furthermore, the low expression of Gfat impacted on chitin biosynthesis, therefore the new cuticle thickening was obstructed.3. Construction of recombinant prokaryotic expression plasmids of CYP303A1 and CPRAfter isolation of CPR cDNA full length sequence, the prokaryotic expression plasmids was constructed through the strategy of ompA+2 and pelB modification. The advantage of using ompA+2 modification strategy was that the coding region of CYP450 cDNA will not be altered, and then the bacteria can hydrolyse the leading peptide sequence ompA+2 and pelB through its own proteolytic functions. Therefore, an intact CYP450 and CPR can be released.