›› 2018, Vol. 61 ›› Issue (5): 519-526.doi: 10.16380/j.kcxb.2018.05.001

• 研究论文 •    下一篇

褐飞虱ATP合酶b亚基基因ATPSb的克隆与功能分析

冯娅琳, 郝培应*, 俞飞飞, 陆潮峰, 朱家骏, 俞晓平*   

  1. (中国计量大学生命科学学院, 浙江省生物计量及检验检疫技术重点实验室, 杭州 310018)
  • 出版日期:2018-05-20 发布日期:2018-05-20

Molecular cloning and function analysis of ATP synthase b subunit gene ATPSb in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

FENG Ya-Lin, HAO Pei-Ying*, YU Fei-Fei, LU Chao-Feng, ZHU Jia-Jun, YU Xiao-Ping*   

  1.  (Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China)
  • Online:2018-05-20 Published:2018-05-20

摘要: 【目的】克隆褐飞虱Nilaparvata lugens ATP合酶b亚基基因全长cDNA序列,并通过RNA干扰技术对该基因的功能进行研究,探讨其在褐飞虱防治中的潜在价值。【方法】根据本实验室获得的褐飞虱转录组测序数据提供的ATP合酶b亚基基因部分核心序列信息,以水稻品种TN1饲养的褐飞虱为材料,应用cDNA末端快速扩增技术(RACE)获得全长cDNA;通过实时荧光定量PCR技术研究其在褐飞虱不同发育阶段和5龄若虫不同组织中的表达谱;并利用褐飞虱2龄若虫通过饲喂法对该基因进行RNAi实验。【结果】成功克隆了褐飞虱ATP合酶b亚基基因ATPSb的全长cDNA序列(GenBank登录号: MF973493)。该序列包含一个843 bp的完整开放阅读框,编码280个氨基酸。荧光定量PCR表明,ATPSb基因的mRNA水平在褐飞虱不同发育阶段以1-2龄若虫期为高,随着龄期增大呈下降趋势,在雌成虫中也随着羽化时间增加而下降,在雄成虫中的表达量高于雌成虫。在5龄若虫不同组织中的表达量以胸部中为最高,头部、中肠、卵巢和脂肪体中相对表达量较低。从靶向ATPSb基因的RNA干扰第6天起,处理组中靶标基因的mRNA水平降低,并且RNA干扰对褐飞虱若虫具有显著致死效果;在RNA干扰后第18天时,对照组个体存活率仍保持在约80%的较高水平,而饲喂dsATPSb的RNA干扰组基本无个体存活。【结论】本研究结果显示,ATPSb基因对褐飞虱的生存具有重要作用,靶向ATPSb基因的RNA干扰对褐飞虱具有显著的抑制效果,ATPSb基因可以作为褐飞虱防治的潜在靶标进行深入研究。

关键词: 褐飞虱, ATP合酶, ATPSb; RNA干扰, 存活率

Abstract: 【Aim】 This study aims to investigate the functions of the ATP synthase b subunit gene in the rice brown planthopper (BPH), Nilaparvata lugens by RNAi and to explore the potential using the gene as a target to control this pest. 【Methods】 The full-length cDNA of ATP synthase b subunit gene was cloned from the BPH reared on rice variety TN1 by RACE according to the sequence information available in the transcriptome data previously obtained by our laboratory. The expression profiles of this gene in different developmental stages and different tissues of the 5th instar nymphs of N. lugens were detected with real-time quantitative PCR, and the RNAi experiments of the gene were carried out with the 2nd instar nymphs of N. lugens by using feeding method. 【Results】 The full-length cDNA of the ATP synthase b subunit gene of the BPH named ATPSb (GenBank accession number: MF973493) was successfully cloned. It contains an ORF of 843 bp, encoding a protein of 280 amino acid residues. Real-time quantitative PCR showed that the ATPSb gene was highly expressed in the 1st and 2nd instar nymphs, and its relative expression level decreased with the developmental stage. The mRNA level of ATPSb was higher in male adults than that in female adults. In the 5th instar nymphs, the mRNA level of ATPSb in the thorax was the highest among different tissues, while relatively low in the head, midgut, ovary and fat body. The RNAi results showed that the mRNA level of ATPSb was significantly decreased in the dsATPSb treatment group from the 6th day, and the RNAi treatment led to distinct mortality of the BPH nymphs. At 18 d after RNAi, the survival rate in the control group kept 80%, while no individual survived in the dsATPSb treatment group. 【Conclusion】 The results suggest that the ATPSb gene is essential to the survival of the BPH, and the RNAi of ATPSb shows an effective inhibition of the BPH. ATPSb may serve as a potential target gene for BPH control.

Key words: Nilaparvata lugens, ATP synthase, ATPSb, RNA interference, survival rate