飞蝗表皮蛋白基因LmNCP1的分子特性及功能分析

doi:10.3864/j.issn.0578-1752.2018.07.008

Abstract

Abstract: 【Objective】The objective of this study is to search and clone a NCP1 from Locusta migratoria (LmNCP1) based on transcriptome database, analyze its sequence and expression characteristics. Then its expression was determined after 20- hydroxyecdysone (20E) induction and interference 20E receptor LmEcR in individuals by RNAi, respectively. Meanwhile, the function of LmNCP1 was analyzed by H&E staining based on RNAi with dsLmNCP1. The results will provide a theoretical basis for the pest control.【Method】A cuticle protein gene was obtained according to the transcriptome database of locusts, and the length of the cDNA sequence was cloned by reverse-transcription PCR (RT-PCR) and sequenced. The gene structure and sequence characteristics were analyzed by using the genome sequence of the locusts. Using the MEGA 6.0 software, the neighbor-joining (NJ) method was used to construct evolutionary tree with the homologous sequences of NCP1 from other insects. Expression profiles of LmNCP1 at different developmental days and in different tissues at day 2 of 5th instar nymphs were assayed using reverse-transcription quantitative PCR (RT-qPCR). Using RT-qPCR, the expression of LmNCP1 was detected after treated with 20E in vivo for 1, 3, 6, 12 h and interfering with 20E receptor gene LmEcR by RNAi for 48 and 72 h, respectively. The biological function of LmNCP1 was analyzed by combination of RNAi and H&E staining method. 【Result】 A cuticle protein gene was obtained by searching the transcriptome database of L. migratoria. The length of the cDNA is 457 bp and full length of its ORF is 306 bp, encoding 101 amino acids. Amino acid sequence analysis showed that the protein encoded by the gene contains one signal peptide and three repeated motifs, but no chitin binding domain, and the repeating motifs were conserved among species by Weblogo analysis. Phylogenetic tree analysis showed that the protein has a close genetic relationship with BcNCP1 of Blaberus craniifer. Thus, the protein was named as LmNCP1 (GenBank accession number: MF326211) according to the result of phylogenetic tree. The results of RT-qPCR showed that LmNCP1 was highly expressed in integument at day 2 of 5th instar nymphs, and lower expression in wing pads, foregut and fat body, but low or no expression in other tested tissues. The expression of LmNCP1 was high at the early stages (N5D1 and N5D2) of 5th instar nymphs, and then decreased (N5D3-N5D6), following a increase at before ecdysis of next instar (N5D7), which is coincidence with the formation of endocuticle and exocuticle. Compared with the control group, the expression of LmNCP1 significantly increased by 1.3 and 0.9 times after injection with 20E for 1 h and 3 h. After 48 h and 72 h of LmEcR interference by RNAi, the expression of LmNCP1 significantly decreased by 71% and 87%, respectively, compared with the control group. After declining the expression of LmNCP1 by RNAi at day 2 of 4th and 5th instar nymphs, the locusts could normally molt and no obvious phenotype was observed. However, the endocuticle of the locusts was thinner than that of the control by H&E staining. 【Conclusion】 A cuticle protein gene LmNCP1 was obtained according to the transcriptome database of L. migratoria. The protein encoded by LmNCP1 does not contain chitin binding domain, but contains three repeated motifs. LmNCP1 was mainly expressed in integument among all the tested tissues. Furthermore, LmNCP1 responds to the regulation of LmEcR-mediated 20E signaling pathway. The results of RNAi suggested that LmNCP1 was involved in the formation of endocuticle during L. migratoria molting.

Key words: Locusta migratoria, cuticle protein, LmNCP1, 20-hydroxyecdysone (20E)

YANG YaTing, ZHAO XiaoMing, QIN ZhongYu, LIU WeiMin, MA EnBo, ZHANG JianZhen. Molecular Characteristics and Function Analysis of Cuticle Protein Gene LmNCP1 in Locusta migratoria[J].Scientia Agricultura Sinica, 2018, 51(7): 1303-1314.

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