Cytochrome b5(Cyt-b5)is a small heme protein and known to be involved in a wide range of biochemical transformations,in eluding cytochrome P450 monooxyge nase(CYP)-mediated metabolism of endoge nous and exogenous compo un ds.Studies on Cyt-b5 are more con centrated in mammals,but are relatively rare in in sects.The characteristics and functi on of Cyt-b5 from Locusta migratoria have not been described yet.We sequeneed the full-length cDNA sequenee of Cyt-b5 from L.migratoria(LmCyt-b5)by reverse transcription-PCR(RT-PCR)based on locust transcriptome database.The phylogenetic analysis showed that LmCyt-b5 was closely related to the Cyt-b5 from Blattodea.LmCyt-b5 was highly expressed in ovary,Malpighian tubules,midgut,gastric caeca,and fat bodies.Silencing of LmCyt-b5 had no effect on the susceptibility of L.migratoria to four different insecticides.Suppression of LmCyt-b5 or silencing of both LmCyt-b5 and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin(7-EC).However,coexpression of LmCYP6FD1 with LmCPR and LmCyt-b5 together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of L.mCYP6FD1 with cytochrome P450 reductase(LmCPR)or LmCyt-b5 separately.These results suggest that LmCyt-b5 plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments.Further study is needed to clarify the role of LmCyt-b5 in CYP-mediated catalytic reactions in L.migratoria.
LIU JiaoZHANG Xue-yaoWU Hai-huaMA WenZHU Wen-yaKun-Yan ZHUMA En-boZHANG Jian-zhen
Cytochrome P450 monooxygenases(CYPs)play essential physiological functions in insects.CYP303A1 is highly conserved in insect species studied to date,and shows an indispensable role for adult eclosion in both Locusta migratoria and Drosophila melanogaster.However,how CYP303A1 is regulated to control insect developmental processes remains uninvestigated.In this study,we discovered functional binding sites for miR-184 in the coding sequence of LmCYP303A1.The luciferase reporter assay showed that miR-184 could target LmCYP303A1 and regulate its expression in vitro.Furthermore,overexpression of miR-184 through microinjection of agomir to locusts reduced the transcripts of LmCYP303A1 and led to abnormal molting,which is similar to the phenotype of silencing LmCYP303A1 by direct injection of dsLmCYP303A1 to locusts.Meanwhile,down-regulation of miR-184 by injection of antagomir increased the LmCYP303A1 transcript and caused molting defects.These findings suggested that miR-184 could target LmCYP303A1 to regulate the molting process in L.migratoria,which might be considered as a novel target for pest control.
Halloween genes are involved in the biosynthesis of the molting hormone, which plays a key role in insect ecdysis, development, metamorphosis, and reproduction. Our previous work identified five Halloween genes from Locusta migratoria, but their functions are currently unknown. In this study, the sequences of these five Halloween genes were analyzed and characterized. LmCYP307a2, LmCYP306a1, LmCYP302a1, and LmCYP315a1 were primarily expressed in the prothoracic glands, while LmCYP314a1 was universally expressed in peripheral tissues, especially in the ovaries and Malpighian tubules. All five Halloween genes were mainly expressed from the 5th to the 7th d in 5th-instar nymphs. RNA interference (RNAi) silencing of LmCYP307a2 resulted in severe molting delays and molting failure, which could be rescued by supplementary 20-hydroxyecdysone. A hematoxylin and eosin staining analysis suggested that the RNAi of LmCYP307a2 inhibited the ecdysis process by inhibiting the apolysis and degradation of the old cuticle, and by promoting the synthesis of a new cuticle. Quantitative reverse transcription polymerase chain reaction results showed that the expressions of LmE74, LmCht5, and LmCht10 were dramatically down-regulated, while that of LmChsI was substantially up-regulated, after knockdown of LmCYP307a2. The results suggest that LmCYP307a2 is related to the molt process via regulation of chitin synthesis and degradation.
Xue-Yao ZhangQi-Hui HeTing-Ting ZhangHai-Hua WuJian-Zhen ZhangEn-Bo Ma
