Knockdown of a putative Halloween gene Shade reveals its role in ecdysteroidogenesis in the small brown planthopper Laodelphax striatellus.
Ecdysteroid hormone 20-hydroxyecdysone (20E) plays fundamental roles in insect development and reproduction, whereas the primary role of ecdysone (E) is the precursor for 20E. A cytochrome P450 monooxygenase (CYP), encoded by a Halloween gene Shade (Shd, cyp314a1), catalyzes the conversion of E into 20E in representative insect species in Diptera, Lepidoptera and Orthoptera. We describe here the cloning and characterization of LsShd in a hemipteran insect species, the small brown planthopper Laodelphax striatellus. LsSHD has five insect conserved P450 motifs, i.e., Helix-C, Helix-I, Helix-K, PERF and heme-binding motifs. Temporal expression pattern of LsShd was determined through the fourth-instar and the early fifth-instar stages by qPCR. LsShd showed two expression peaks in day 2 and day 5 fourth-instar nymphs, and two troughs in day 1 fourth and fifth instars. Dietary introduction of double-stranded RNA (dsRNA) of LsShd into nymphs successfully knocked down the target gene, decreased expression level of ecdysone receptor (LsEcR) gene, and caused nymphal lethality and delayed development. Ingestion of 20E did not increase LsShd expression level, but almost completely rescued LsEcR mRNA level, and relieved the negative effects on the survival and development in LsShd-dsRNA-exposed nymphs. In contrast, dietary introduction of E had little rescue effects. Thus, our data suggest that the ecdysteroidogenic pathway is conserved in insects, and LsSHD functions to regulate metamorphotic processes by converting E to 20E even in a hemipteran insect, L. striatellus.
Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
Shuang
S
Jia
Pin-Jun
PJ
Wan
Li-Tao
LT
Zhou
Li-Li
LL
Mu
Guo-Qing
GQ
Li
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004440:Ecdysone / Q000096:biosynthesis / Q000378:metabolism; D026461:Ecdysteroids / Q000096:biosynthesis*; D004441:Ecdysterone / Q000096:biosynthesis; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D055785:Gene Knockdown Techniques; D017344:Genes, Insect / Q000235:genetics / Q000502:physiology*; D006430:Hemiptera / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D008675:Metamorphosis, Biological / Q000235:genetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D026461:Ecdysteroids; D004440:Ecdysone; D004441:Ecdysterone; D003577:Cytochrome P-450 Enzyme System
20-hydroxyecdysone; 20E; ANOVA; CYP; E; EcR; Ecdysteroidogenesis; Laodelphax striatellus; ML; ORF; PCR; Performance; RNA interference; RNAi; RT-PCR; SE; Shade; YLS; analysis of variance; cDNA; complementary DNA; cytochrome P450 monooxygenase; double-stranded RNA; dsRNA; ecdysone; ecdysone receptor; maximum-likelihood; open reading frame; polymerase chain reaction; qPCR; quantitative real-time PCR; reverse transcriptase PCR; standard error; yeast-like symbionts