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(Gen Bank登录号:KM217014.1),NlCyp306a1(Gen Bank登录号:KM217013.1),NlCyp302a1(Gen Bank登录号:KM216995.1),NlCyp315a1(Gen Bank登录号:KM216998.1)和NlCyp314a1(Gen Bank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。Nl CYP314A1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
【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
作者
李凯龙
傅强
王渭霞
赖凤香
万品俊
LI Kai-Long;FU Qiang;WANG Wei-Xia;LAI Feng-Xiang;WAN Pin-Jun(State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, China;Hunan Provincial Center for Monitoring of Grain Oil Products Quality, Changsha 410201, China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2017年第10期1129-1140,共12页
Acta Entomologica Sinica
基金
浙江省自然科学基金(Y15C140023)
现代农业产业技术体系建设专项基金资助项目(CARS-01-18)
