Cloning And Characterization Of Halloween Genes Involving In Ecdysteriodogenesis In Sogatella Furcifera And Laodelphgax Striatellus

In insects, ecdysteroid hormones ecdysone and20-hydroxyecdysone play fundamental roles in insect postembryonic development and reproduction. Ecdysone is synthesized from precursor cholesterol in the prothoracic glands, a pair of endocrine organs located in the insect’s pro thorax, or the homologs such as dipteran ring glands. Five cytochrome P450monooxygenases (CYPs), encoded by Halloween genes, are involved in the ecdysteroidogenesis. These CYP enzymes are characterized only in several insect species. RNA interference (RNAi) technology can be used to silence the target gene to study gene function. Therefore, introduction dsRNA into insects to knockdown target gene expression to explore gene function has become a widely used method.The white-backed planthopper Sogatella furcifera (Horvath) and the small brown planthopper Laodelphgax striatellus (Fallen) are two important rice pests in China. In this study, based on the transcriptome data, RT-PCR and RACE, we identified five genes encoding cytochrome P450monooxygenase enzymes involve in ecdysteroidogenesis in S. furcifera and L. striatellus. Moreover, we used RNAi-mediated gene knockdown to test the effects of these proteins on nymphal survival, growth and development. The main results were listed as follows.1. Identification of Halloween genesBy RACE, the cDNAs of Halloween genes in S. furcifera and L. striatellus were cloned and named as SfCYP306A, LsCYP306A, SfCYP302A1, LsCYP302A1, SfCYP314A1, LsCYP314A, SfCYP307A, LsCYP307A, SfCYP315A1and LsCYP315A1seperately.SfCYP306A, LsCYP306A, SfCYP302A1, LsCYP302A1LsCYP314A1and LsCYP315A1have five insect conserved P450motifs, i.e., WxxxR (Helix-C), GxE/DTT/S (Helix-I), ExxR (Helix-K), PxxFxPE/DRF (PERF motif) and PFxxGxRxCxG/A (heme-binding domain). In case of SfCYP315A1, however, two conserved C terminal motifs, PERF motif and hemi-binding domain, were missed. For SfCYP307A1and LsCYP307A1, Helix-C and Helix-I are not conserved, but the sequences of putative regions on these motifs are very similar in insects. Unrooted phylogenetic tree clearly separated into CYP2clan (CYP307A and CYP306A) and mitochondrial clan (CYP302A1, CYP315A1and CYP314A1). The former clan was separated to CYP307A and CYP306A clades. The latter clan was subdivided into CYP315A1, CYP302A1and CYP314A1clades.2. Stage-specific expression profileThe expression profile of all the five Halloween genes in4th-instar and the early5th-instar nymphs in both rice planthoppers was determined by quantitative real-time PCR. In S. furcifera, these five genes were highly expressed in the1st-day4th-instar nymphs. In the2nd-and3rd-day after ecdysis, the expression levels were dramatically decreased. In newly-molted5th-instar nymphs, the expression levels were sharply increased. In L. striatellus, all the five Halloween genes showed two expression peaks in the2nd-and5th-day4th-instar nymphs. In newly-molted4th-and5th-instar nymphs, the expression levels were lower and formed two troughs. Accordingly, it can be speculated that the temporal expression pattern of the five Halloween genes in S. furcifera and L. striatellus should coincide with its ecdysteroid titer in the haemolymph.3. RNAi-mediated characterization of the five Halloween genesSeveral liquid diets contained dsCyp306a1, dsCyp307a1, dsCyp302a1,dsCyp314a1and dsCyp315a1at the concentration of0.5mg/ml were prepared. The2nd-instar nymphs of S. furcifera and the4th-instar nymphs of L. striatellus were continuously exposed to each of the diet for6days and5days respectively. The mRNA abundance of the five Halloween genes in the surviving nymphs was examined by quantitative real-time PCR (qPCR). RNAi-mediated knockdown of each of the five Halloween genes reduced the expression of target gene and LsEcR at the mRNA level, caused nymphal lethality, and developmental delay. Moreover,20E application almost completely rescued EcR expression in S. furcifera and L. striatellus nymphs that ingested dscyp307a1. Since the expression of EcR gene was regulated by ecdysteroids through a positive feedback loop, the down-regulation of the EcR expression should result from a considerable decrease in the ecdysteroid titer in S. furcifera and L. striatellus haemolymph caused by dsRNA ingestion. Thus, our results suggest that the ecdysteroidogenic pathway was conserved among insect species, and the Halloween genes are responsible for specific steps in the synthesis of ecdysteroid in S. furcifera and L. striatellus.