中国农业科学 ›› 2015, Vol. 48 ›› Issue (15): 2976-2984.doi: 10.3864/j.issn.0578-1752.2015.15.007

• 植物保护 • 上一篇    下一篇

褐飞虱Yellow基因的克隆及功能

王博,姚云,徐泽炜,林欣大   

  1. 中国计量学院生命科学学院,杭州 310018
  • 收稿日期:2015-02-16 出版日期:2015-08-01 发布日期:2015-08-01
  • 通讯作者: 林欣大,E-mail:linxinda@cjlu.edu.cn E-mail:linxinda@cjlu.edu.cn
  • 作者简介:王博,E-mail:824419396@qq.com
  • 基金资助:
    国家自然科学基金面上项目(31471771)

Molecular Cloning and Function of the Yellow Gene from Nilaparvata lugens

WANG Bo, YAO Yun, XU Ze-wei, LIN Xin-da   

  1. College of Life Sciences, China Jiliang University, Hangzhou 310018
  • Received:2015-02-16 Online:2015-08-01 Published:2015-08-01

摘要: 【目的】克隆褐飞虱(Nilaparvata lugens)Yellow基因(NlYellow),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NlYellow了解其功能。【方法】使用网络版primer3设计引物克隆NlYellow,将得到的cDNA序列翻译为氨基酸序列后,与GenBank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(qRT-PCR)技术,研究褐飞虱胚胎、1-5龄若虫以及成虫期NlYellow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0.4 µg针对NlYellow的双链RNA,待羽化为成虫后观察表型。【结果】克隆得到NlYellowORF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。qRT-PCR显示NlYellow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,NlYellow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NlYellow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NlYellow的功能是参与外表皮色素沉积,改变体色。

关键词: 褐飞虱, 黄体基因, 基因表达谱, RNA干扰

Abstract: 【Objective】The objectives of this study are to clone NlYellow 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 NlYellow 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 GenBank. Also a Neighbor-Joining method was used to construct a phylogenetic tree. Quantitative real-time PCR (qRT-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). NlYellow dsRNA at 0.4 µg was injected into 5th instar nymphs and the adult phenotypes were observed. 【Result】The NlYellow was cloned, and through sequence alignment it was found that the NlYellow was more similar to the ApYellow (98%), but it was less conserved when compared with that of Drosophila melanogaster and Bombyx mori.Phylogenetic analysis showed that it clustered with Acyrthosiphon pisum,too. NlYellow’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 NlYellow 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, NlYellow 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<0.05). Using RNA interference to knock down the NlYellow expression, it was found that the color of the whole body turned into yellow, especially in thorax, abdomen and legs.【Conclusion】NlYellow might involved in insect pigmentation and change of the body colour.

Key words: Nilaparvata lugens; Yellow, gene expression profile, RNA interference

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