关键词: 褐飞虱, 核受体; NlE75, 转录本, RNAi, 蜕皮

Abstract: 【Aim】 Ecdysone regulates the molting and metamorphosis processes of insects through signaling cascade, and the nuclear receptor gene E75 is an early response gene of the signaling pathway. The objective of this study is to analyze the molecular characteristics and biological function of NlE75 in the brown planthopper, Nilaparvata lugens, so as to provide new molecular target for the management of N. lugens and development of new pesticides. 【Methods】 The cDNA sequence of NlE75 was obtained based on sequence alignment, homology search and N. lugens transcriptome data, and cloned by RT-PCR and RACE technologies. The structure characteristics of the protein encoded by NlE75 were predicted using bioinformatics software, and the phylogenetic tree was constructed using the neighbor-joining method of MEGA 5.0. The expression profiles of NlE75 and its transcripts in different developmental stages (egg, 1st－5th instar nymphs and adult), different tissues (head, thorax, abdomen, cuticle, wing bud, fat body, midgut and leg) of the 5th instar nymph, and the 3rd instar nymph of N. lugens after injection of 0.2 μg/individual of 20-hydroxyecdysone (20E) were detected by RT-qPCR. After knockdown of NlCYP314A1 in the 3rd instar nymph by RNAi via dsRNA injection, the expression level of NlE75 in the 3rd instar nymphs was detected by RT-qPCR. After downregulating the expression of NlE75 in the 3rd and 5th instar nymphs and its transcripts in the 3rd instar nymphs by RNAi via dsRNA injection, the expression levels of NlE75 in the 3rd instar nymphs were detected by RT-qPCR, the effects of knockdown of NlE75 on the molting and metamorphosis of the 3rd and 5th instar nymphs of N. lugens were observed, and the regulatory relationships of five transcripts of NlE75 were analyzed. 【Results】 Five NlE75 transcripts NlE75A, NlE75B, NlE75C, NlE75D and NlE75E (GenBank accession numbers are ON086330, ON086331, ON086332, ON086333 and ON086334, respectively) of N. lugens were cloned, and their encoded proteins all have typical nuclear receptor conserved domains. Phylogenetic analysis revealed that NlE75A-E clustered with the E75 proteins of Pediculus humanus corporis and Blattella germanica. RT-qPCR results showed that five NlE75 transcripts had different developmental and tissue expression characteristics. The expression levels of NlE75A and NlE75D in various developmental stages of N. lugens were low, the expression dynamics of NlE75A, NlE75B and NlE75C were consistent with that of NlE75 and the expression level of NlE75D increased to the highest level before the molting of both the 4th and 5th instar nymphs, while the expression levels of NlE75E in adults were the highest. The five NlE75 transcripts were expressed in various tissues of the 5th instar nymphs, with NlE75B, NlE75C, NlE75D and NlE75E highly expressed in the leg, and NlE75A highly expressed in the fat body. The expression levels of NlE75 in treatment groups significantly increased by 4.7 and 5.0 times, respectively, at 4 and 8 h after 20E induction, as compared to those in the corresponding control groups. The expression level of NlE75 in treatment group was extremely significantly down-regulated by 47.8% at 48 h after RNAi of NlCYP314A1, as compared with that in the control group (injected with dsGFP). RNAi results showed that injection of dsNlE75 into the 3rd instar nymphs resulted in the failure of molting and phenotypic defects, while injection of dsNlE75 into the 5th instar nymphs resulted in the failure of metamorphosis and phenotypic defects, and the 3rd and 5th instar nymphs injected with dsNlE75 could not complete the molting process and eventually died 100%. The phenotypic defects appeared only when all the five transcripts of NlE75 were knocked down, but did not appear when a single transcript was knocked down, suggesting that there is a complex regulatory relationship between the five transcripts. 【Conclusion】 There are five NlE75 transcripts in N. lugens, with different developmental and tissue expression characteristics. RNAi results suggest that five NlE75 transcripts have obvious functional redundancy and NlE75 plays an important role in the molting of N. lugens.

Key words: Nilaparvata lugens, nuclear receptor, NlE75, transcript, RNAi, molting