褐飞虱Yellow基因的克隆及功能  

Molecular Cloning and Function of the Yellow Gene from Nilaparvata lugens

作　　者：王博[1] 姚云[1] 徐泽炜 林欣大[1] 

机构地区：[1]中国计量学院生命科学学院,杭州310018

出　　处：《中国农业科学》2015年第15期2976-2984,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31471771)

摘　　要：【目的】克隆褐飞虱(Nilaparvata lugens)Yellow基因(Nl Yellow),研究该基因在褐飞虱不同发育时期和不同组织中的表达谱,通过RNA干扰沉默Nl Yellow了解其功能。【方法】使用网络版primer3设计引物克隆Nl Yellow,将得到的c DNA序列翻译为氨基酸序列后,与Gen Bank数据库中其他物种的Yellow序列进行同源比对,并构建系统发育树。采用实时荧光定量PCR(q RT-PCR)技术,研究褐飞虱胚胎、1-5龄若虫以及成虫期Nl Yellow的相对表达量,并检测该基因在雌、雄成虫头、胸、腹、足、翅、卵巢和睾丸等组织中的相对表达量。向褐飞虱5龄若虫体内注射0.4μg针对Nl Yellow的双链RNA,待羽化为成虫后观察表型。【结果】克隆得到Nl Yellow ORF序列,通过序列比对发现它与豌豆蚜(Acyrthosiphon pisum)Yellow的氨基酸序列同源性最高(98%),但与黑腹果蝇(Drosophila melanogaster)、家蚕(Bombyx mori)等物种的氨基酸序列同源性较低。系统发育树分析显示它与豌豆蚜的亲缘关系最近。q RT-PCR显示Nl Yellow在胚胎期表达水平具有波动性,其中第1、3、4和5天中的表达量低于其他时段的表达量。此外,Nl Yellow在3龄和5龄若虫期表达量高于其他若虫期(P<0.05);该基因在雄虫头、胸、翅、足、中肠和睾丸中均有表达,但前4个组织中表达量相对较高(P<0.05);而在雌虫中,该基因只在头、胸、翅、足中表达。另外,该基因在短翅成虫中表达水平显著高于长翅成虫(P<0.05),并且在短翅成虫当中,雄虫中的表达量高于雌虫并达到显著水平(P<0.05)。采用RNA干扰技术沉默Nl Yellow后,褐飞虱成虫整体体色变黄,胸、腹和足颜色变化尤其明显。【结论】初步推测Nl Yellow的功能是参与外表皮色素沉积,改变体色。【Objective】The objectives of this study are to clone NlY ellow from the brown planthopper（Nilaparvata lugens）, explore the expression profiles during the development in adults and in different tissues, then study its function through RNA interference.【Method】Primers were designed by online version of primer 3 software and NlY ellow was cloned, then the nucleotide sequence was translated into amino acid sequence. The alignment of amino acids residues was conducted using N. lugens Yellow and others searched from Gen Bank. Also a Neighbor-Joining method was used to construct a phylogenetic tree. Quantitative real-time PCR（qR T-PCR） was applied to measure the relative expression levels of N. lugens at different developmental stages（embryo, 1st-5th instar nymphs and adult） and in different tissues（head, thorax, abdomen, leg, wing, midgut, ovary, and testis）. NlY ellow dsR NA at 0.4 μg was injected into 5th instar nymphs and the adult phenotypes were observed. 【Result】The NlY ellow was cloned, and through sequence alignment it was found that the NlY ellow was more similar to the ApY ellow（98%）, but it was less conserved when compared with that of Drosophila melanogaster and Bombyx mori. Phylogenetic analysis showed that it clustered withAcyrthosiphon pisum, too. NlY ellow’s expression was variable in the embryo, the expression level in 1st, 3rd, 4th and 5th day were lower than other days. Furthermore, its expression levels were higher in 3rd and 5th instar nymph than others（P〈0.05）. Tissue-specific expression analysis elucidated that NlY ellow expressed in all tissues including head, thorax, abdomen, leg, wing, midgut and testis in male, but the first four tissues had higher expression level（P〈0.05）. On the contrary, NlY ellow was only expressed in head, thorax, leg, and wing in female. Furthermore, its expression level in short wing adult was higher than that of long wing adult（P〈0.05）. Also in short wing adults, male’s expression level was higher than female（P�

关 键 词：褐飞虱 黄体基因 基因表达谱 RNA干扰 

分 类 号：S433[农业科学—农业昆虫与害虫防治]