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The P 450 enzyme S hade mediates the hydroxylation of ecdysone to 20‐hydroxyecdysone in the C olorado potato beetle, L eptinotarsa decemlineata
Author(s) -
Kong Y.,
Liu XP.,
Wan PJ.,
Shi XQ.,
Guo WC.,
Li GQ.
Publication year - 2014
Publication title -
insect molecular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.955
H-Index - 93
eISSN - 1365-2583
pISSN - 0962-1075
DOI - 10.1111/imb.12115
Subject(s) - biology , ecdysone receptor , 20 hydroxyecdysone , spodoptera , ecdysone , colorado potato beetle , ecdysteroid , sf9 , microbiology and biotechnology , biochemistry , botany , nuclear receptor , gene , larva , recombinant dna , transcription factor
Ecdysone 20‐monooxygenase ( E20MO ), a cytochrome P450 monooxygenase ( CYP314A1 ), catalyses the conversion of ecdysone ( E ) to 20‐hydroxyecdysone ( 20E ). We report here the cloning and characterization of the H alloween gene S hade ( S hd ) encoding E20MO in the C olorado potato beetle, L eptinotarsa decemlineata . LdSHD has five conserved motifs typical of insect P450s , ie the H elix‐ C , H elix‐ I , H elix‐ K , P xx F x PE/DRF ( PERF ) and heme‐binding motifs. LdShd was expressed in developing eggs, the first to fourth instars, wandering larvae, pupae and adults, with statistically significant fluctuations. Its mRNA was ubiquitously distributed in the head, thorax and abdomen. The recombinant LdSHD protein expressed in S podoptera frugiperda 9 ( Sf9) cells catalysed the conversion of E to 20E . Dietary introduction of double‐stranded RNA ( dsRNA ) of LdShd into the second instar larvae successfully knocked down the LdShd expression level, decreased the mRNA level of the ecdysone receptor ( LdEcR ) gene, caused larval lethality, delayed development and affected pupation. Moreover, ingestion of LdShd ‐ dsRNA by the fourth instars also down‐regulated LdShd and LdEcR expression, reduced the 20E titre, and negatively influenced pupation. Introduction of 20E and a nonsteroidal ecdysteroid agonist halofenozide into the LdShd ‐ dsRNA ‐ingested second instars, and of halofenozide into the LdShd ‐ dsRNA ‐ingested fourth instars almost completely relieved the negative effects on larval performance. Thus, LdSHD functions to regulate metamorphotic processes by converting E to 20E in a coleopteran insect species Le . decemlineata .