褐飞虱蜕皮激素合成相关Halloween基因的克隆及功能分析

doi:10.16380/j.kcxb.2017.10.004

摘要/Abstract

摘要： 【目的】蜕皮激素（ecdysone）是调控昆虫生长发育和繁殖的重要激素，其合成主要由5个称为Halloween基因编码的细胞色素P450（spook/CYP307A1, Phantom/CYP306A1, disembodied/CYP302A1, shadow/CYP315A1和shade/CYP314A1）参与。褐飞虱Nilaparvata lugens系东南亚和东亚地区危害最为严重的水稻害虫之一，以蜕皮激素调控基因为靶标，应用RNAi技术防控褐飞虱正逐步成为研究热点。本研究旨在揭示褐飞虱体内Halloween基因的功能，为褐飞虱的防控和新农药的开发提供理论依据。【方法】本研究基于褐飞虱基因组和转录组数据，利用RT-PCR技术克隆了参与蜕皮激素合成的5个Halloween 基因；利用生物信息学软件预测分析其编码蛋白的结构特性；利用MEGA5.0邻接法构建系统进化树；利用实时荧光定量PCR技术研究其时空表达特征；利用注射法RNAi技术分析Cyp314a1基因表达下调对褐飞虱生长发育和繁殖的影响。【结果】克隆获得褐飞虱的5个Halloween基因，即NlCyp307a1（GenBank登录号: KM217014.1）, NlCyp306a1（GenBank登录号: KM217013.1）, NlCyp302a1（GenBank登录号: KM216995.1）, NlCyp315a1（GenBank登录号: KM216998.1）和NlCyp314a1（GenBank登录号: KU928172）。进化分析表明，这5个基因分属于CYP2和线粒体两大类群，并与各自的同源基因聚类在一起，说明Halloween基因和蜕皮激素合成途径在昆虫进化过程中十分保守。NlCYP314A1具有5个P450的保守结构域和两个跨膜结构域，是典型的线粒体酶。qRT-PCR检测结果显示，5个Halloween基因的表达量在整个褐飞虱5龄若虫期内呈现波动状，且均在5龄若虫蜕皮后的24 h和60 h出现表达量峰值。NlCyp314a1在褐飞虱发育各个龄期均表达，且在成虫中表达量最高；NlCyp314a1在胸部表达量较头部和腹部高，其在脂肪体中的表达量最高，其次是在足、表皮、翅芽中，中肠中表达量最低。利用注射法RNAi对NlCyp314a1基因进行沉默后，NlCyp314a1的表达量在注射后第4天较对照极显著降低84.6%（P<0.01），同时蜕皮激素通路下游的响应关键基因NlFTZF1的表达量较对照极显著降低64.1%（P<0.01）。表型观察发现，NlCyp314a1表达量的下调导致了褐飞虱蜕皮困难，出现畸形虫体，第7天死亡率大于95%，且NlCyp314a1表达量的下调导致了褐飞虱卵巢发育畸形。【结论】褐飞虱蜕皮激素合成相关的Halloween基因在进化上非常保守，Halloween基因功能的研究揭示了褐飞虱体内Halloween基因对于褐飞虱蜕皮和繁殖的重要性。其表达水平的降低导致了褐飞虱生长发育和繁殖受阻，提示褐飞虱Halloween基因可用于褐飞虱蜕皮和繁殖进程的调控，同样也为Halloween基因在褐飞虱防治方面的潜在应用提供了依据。

关键词: 褐飞虱, 蜕皮激素； Halloween基因, 基因表达, 蜕皮, 繁殖

Abstract: 【Aim】 As one of the most important insects endohormones, ecdysone plays an important role in regulating the development and reproduction of insects. Five Halloween genes are involved in ecdysteroid biosynthesis and encode cytochrome P450 enzymes spook/CYP307A1, Phantom/CYP306A1, disembodied/CYP302A1, shadow/CYP315A1 and shade/CYP314A1, respectively. The brown planthopper, Nilaparvata lugens, is one of the most destructive pests of rice throughout Southeastern and Eastern Asia. For N. lugens, ecdysteroid regulated genes of RNAi technology management has gradually become a research hotspot. The objective of this study is to investigate the function of Halloween genes in N. lugens so as to provide the theoretical basis for the management of N. lugens and development of new pesticides. 【Methods】 Based on the genome and transcriptome database of N. lugens, five ecdysteroidogenesis-related Halloween genes in N. lugens were cloned using RT-PCR, and the deduced protein structure was predicted using different bioinformatics software, and the phylogenetic tree was constructed using the neighbor-joining method of MEGA 5.0. The expression profiles of these genes in different tissues of nymph and at different developmental stages of N. lugens were detected by quantitative real-time PCR. RNAi experiment was carried out to examine the effects of knockdown of Halloween genes on the development and reproduction of N. lugens. 【Results】 Five Halloween genes involved in ecdysteroidogenesis, i.e., NlCyp307a1, NlCyp306a1, NlCyp302a1, NlCyp315a1, and NlCyp314a1, were cloned and identified in N. lugens. Phylogenetic analysis revealed that the five Halloween genes belong to two superfamilies 2-Clan and Mito-Clan, respectively, and clustered with their homologous genes in other insects, suggesting that Halloween genes and edysteroidogenesis pathways are very highly conserved during insect evolution. NlCYP314A1 contains five conserved P450s motifs and two transmembrane domains, being a typical mitochondria cytochrome P450s. The results of qRT-PCR showed that five Halloween genes had higher mRNA abundance at 24 h and 60 h after molting in the 5th instar nymph stage. NlCyp314a1 was continually expressed during the whole developmental stage, and showed the highest expression level in adults. Among the examined tissues, thorax had the higher transcription level of NlCyp314a1 than head and abdomen, and fat body had the highest transcription level of NlCyp314a1, followed by leg, integument, wing bud, and midgut. Four days after injection of dsNlCyp314a1, the expression levels of NlCyp314a1 and NlFTZ-F1 in the treated individuals were extremely significantly decreased by 84.6% and 64.1%, respectively, compared with the control. NlCyp314a1 knockdown resulted in nymphs failed to molt on time, exhibited apparent phenotypic defects, and >95% mortality of the nymphs at day 7 after injection. NlCyp314a1 knockdown also resulted in the developmental abnormality of adult ovary. 【Conclusion】 Halloween genes of N. lugens involving in ecdysteroidogenesis are evolutionarily conserved. Silencing of selected Halloween gene expression causes phenotypic defects in molting and reproduction. These results suggest that Halloween genes encode functional proteins that are involved in molting and reproduction of N. lugens. Halloween genes might be used in altering genetically the progress of molting and reproduction of N. lugens. This study provides a theoretical basis for potential application of Halloween genes in the management of N. lugens.

Key words: Nilaparvata lugens; ecdysone, Halloween gene, gene expression, molting, reproduction

李凯龙, 傅强, 王渭霞, 赖凤香, 万品俊. 褐飞虱蜕皮激素合成相关Halloween基因的克隆及功能分析[J]. , 2017, 60(10): 1129-1140.

LI Kai-Long, FU Qiang, WANG Wei-Xia, LAI Feng-Xiang, WAN Pin-Jun. Molecular cloning and functional characterization of Halloween genes involved in ecdysteroid biosynthesis in the brown planthopper,Nilaparvata lugens (Hemiptera: Delphacidae)[J]. , 2017, 60(10): 1129-1140.

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褐飞虱蜕皮激素合成相关Halloween基因的克隆及功能分析

Molecular cloning and functional characterization of Halloween genes involved in ecdysteroid biosynthesis in the brown planthopper,Nilaparvata lugens (Hemiptera: Delphacidae)

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