飞蝗表皮蛋白基因LmNCP1的分子特性及功能分析

Molecular Characteristics and Function Analysis of Cuticle Protein Gene LmNCP1 in Locusta migratoria

【目的】基于飞蝗(Locusta migratoria)转录组数据库搜索并克隆获得飞蝗表皮蛋白基因Lm NCP1(nymph cuticle protein 1),分析其序列特征和表达特性,通过蜕皮激素(20-hydroxyecdysone,20E)诱导和干扰20E受体基因Lm Ec R表达研究其表达调控,并基于RNA干扰(RNAi)方法分析其生物学功能,为阐明该基因在飞蝗表皮发育过程中的作用提供理论依据。【方法】依据飞蝗转录组数据库获得表皮蛋白基因Lm NCP1,结合RT-PCR技术克隆获得其全长开放阅读框(ORF)序列,并结合飞蝗基因组序列分析其基因结构及序列特征;使用MEGA 6.0软件中邻接法(neighbor-joining,NJ),与其他昆虫同源序列聚类分析,并根据聚类结果进行命名;利用reverse-transcription quantitative PCR(RT-q PCR)分析其在5龄飞蝗不同组织和不同发育天数的基因表达特性;通过体内注射20E诱导以及干扰20E受体基因Lm Ec R的表达,RT-q PCR检测该基因的表达情况;基于RNAi结合H&E染色方法分析其生物学功能。【结果】通过搜索获得表皮蛋白基因Lm NCP1,并进行了克隆和测序验证,获得457 bp的c DNA序列,其全长ORF序列为306 bp,编码101个氨基酸。氨基酸序列分析表明该基因编码的蛋白含有1个信号肽,不含几丁质结合域,但包含3个重复基序,Weblogo分析结果显示其在物种间具有保守性。系统进化树分析显示该蛋白与蜚蠊目蟑螂(Blaberus craniifer)Bc NCP1序列一致度最高,聚为一支。根据聚类结果将该蛋白命名为Lm NCP1(Gen Bank登录号:MF326211)。RT-q PCR结果显示Lm NCP1在5龄若虫体壁中高表达,在翅芽、前肠和脂肪体中表达次之,在中肠、后肠、胃盲囊和马氏管中表达量较低或不表达;Lm NCP1在5龄早期(N5D1和N5D2)高表达,随后表达量逐渐降低(N5D3—N5D6),到下一次蜕皮前表达有所升高(N5D7),其表达量动态与内表皮形成时间一致。20E诱导不同时间结果显示,与对照组相比,Lm NCP1在20E诱导1 h和3 h后表达量显著上调,分别上调了1.3倍和0.9倍;利用RNAi方法干扰Lm Ec R 48 h和72 h后,与对照组相比,Lm NCP1表达量均显著降低,分别降低了71%和87%。利用RNAi方法在4龄和5龄第2天分次注射ds Lm NCP1后,飞蝗仍能够正常蜕皮,无致死表型,但对其表皮进行H&E染色发现,与对照组相比,其内表皮形成减少,导致表皮显著变薄,表皮层厚度约减少了33%。【结论】飞蝗表皮蛋白Lm NCP1含有3个物种间保守的重复基序,不含几丁质结合域,属于其他家族蛋白;Lm NCP1主要在体壁中高表达,而且响应Lm Ec R介导的20E信号通路调控;RNAi试验表明,Lm NCP1在飞蝗蜕皮过程中参与内表皮的沉积。

【Objective】The objective of this study is to search and clone a NCP1 from Locusta migratoria（Lm NCP1） based on transcriptome database, analyze its sequence and expression characteristics. Then its expression was determined after 20-hydroxyecdysone（20 E） induction and interference 20 E receptor Lm Ec R in individuals by RNAi, respectively. Meanwhile, the function of Lm NCP1 was analyzed by HE staining based on RNAi with ds Lm NCP1. 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 c DNA 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 Lm NCP1 at different developmental days and in different tissues at day 2 of 5 th instar nymphs were assayed using reverse-transcription quantitative PCR（RT-q PCR）. Using RT-q PCR, the expression of Lm NCP1 was detected after treated with 20 E in vivo for 1, 3, 6, 12 h and interfering with 20 E receptor gene Lm Ec R by RNAi for 48 and 72 h, respectively. The biological function of Lm NCP1 was analyzed by combination of RNAi and HE staining method. 【Result】 A cuticle protein gene was obtained by searching the transcriptome database of L. migratoria. The length of the c DNA 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 Bc NCP1 of Blaberus craniifer. Thus, the protein was named as Lm NCP1（Gen Bank accession number： MF326211） according to the result of phylogenetic tree. The results of RT-q PCR showed that Lm NCP1 was highly expressed in integument at day 2 of 5 th instar nymphs, and lower expression in wing pads, foregut and fat body, but low or no expression in other tested tissues. The expression of Lm NCP1 was high at the early stages（N5 D1 and N5 D2） of 5 th instar nymphs, and then decreased（N5 D3-N5 D6）, following a increase at before ecdysis of next instar（N5 D7）, which is coincidence with the formation of endocuticle and exocuticle. Compared with the control group, the expression of Lm NCP1 significantly increased by 1.3 and 0.9 times after injection with 20 E for 1 h and 3 h. After 48 h and 72 h of Lm Ec R interference by RNAi, the expression of Lm NCP1 significantly decreased by 71% and 87%, respectively, compared with the control group. After declining the expression of Lm NCP1 by RNAi at day 2 of 4 th and 5 th 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 HE staining. 【Conclusion】 A cuticle protein gene Lm NCP1 was obtained according to the transcriptome database of L. migratoria. The protein encoded by Lm NCP1 does not contain chitin binding domain, but contains three repeated motifs. Lm NCP1 was mainly expressed in integument among all the tested tissues. Furthermore, Lm NCP1 responds to the regulation of Lm Ec R-mediated 20 E signaling pathway. The results of RNAi suggested that Lm NCP1 was involved in the formation of endocuticle during L. migratoria molting.