昆虫学报 ›› 2019, Vol. 62 ›› Issue (3): 294-303.doi: 10.16380/j.kcxb.2019.03.003

• 研究论文 • 上一篇    下一篇

棉铃虫酪氨酸羟化酶基因的分子特性及功能分析

姚雪, 李琳红, 魏纪珍, 席玉强, 杜孟芳, 崔江宽, 安世恒, 刘晓光*   

  1. (河南农业大学植物保护学院, 省部共建小麦玉米作物学国家重点实验室, 郑州 450002)
  • 出版日期:2019-03-20 发布日期:2019-03-14

Molecular characterization and functional analysis of tyrosine hydroxylase gene in Helicoverpa armigera (Lepidoptera: Noctuidae)

YAO Xue, LI LinHong, WEI JiZhen, XI YuQiang, DU MengFang, CUI JiangKuan, AN ShiHeng, LIU XiaoGuang*   

  1. (State Key Laboratory of Wheat and Maize Crop Science, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, China)
  • Online:2019-03-20 Published:2019-03-14

摘要:

【目的】酪氨酸羟化酶(tyrosine hydroxylase, TH)是黑色素形成的关键酶,在昆虫表皮骨化过程中扮演重要角色。本研究旨在获得棉铃虫Helicoverpa armigera TH基因序列,并研究其分子特性、表达模式和功能,为更深入探析该基因作用机理奠定基础。【方法】通过生物信息学和分子生物学技术获得了棉铃虫TH基因序列,利用qRT-PCR分析该基因在棉铃虫不同生长发育阶段的表达模式;利用qRT-PCR技术,分别测定了蜕皮激素20E(400 ng/头)处理不同时间和RNAi成功干扰蜕皮激素受体基因(EcR)前提下再用20E(400 ng/头)处理后,棉铃虫5龄幼虫TH表达量变化;采用生物化学方法检测鞣化激素(30 μg/mg组织)和环腺苷酸(cAMP, 200 ng/mg 组织)处理后棉铃虫幼虫脂肪体中TH活性。【结果】获得了棉铃虫酪氨酸羟化酶基因TH (GenBank登录号: MF440319) cDNA片段,长2 270 bp,开放阅读框1 686 bp,编码561个氨基酸残基。该基因在棉铃虫整个发育期均表达,其中在卵期第3天、2龄幼虫第1天、3-5龄蜕皮期、预蛹期和成虫羽化第1天表达量相对较高。研究还发现,400 ng/头 20E注射能够促进TH的转录;在成功干扰并调低幼虫EcR转录水平的前提条件下(对照仅注射dsGFP)再注射20E,对TH表达量无明显影响;而鞣化激素(30 μg/mg组织)和cAMP(200 ng/mg组织)均显著提高了TH的酶活性。【结论】20E在转录水平参与了TH的表达;鞣化激素和cAMP均能够提高TH活性,在蛋白水平上对TH进行调控。

关键词: 棉铃虫, 酪氨酸羟化酶, RNA干扰, 蜕皮激素, 鞣化激素

Abstract:  【Aim】 Tyrosine hydroxylase (TH) is a key enzyme of melanin biosynthesis and plays an important role in epidermal sclerotization. This study aims to obtain the cDNA sequence of TH gene of Helicoverpa armigera, and to analyze its molecular characteristics, expression pattern and function, so as to provide a foundation for further exploring its action mechanism. 【Methods】 The full-length cDNA sequence of TH gene was cloned from H. armigera by using bioinformatics and molecular cloning technique. The developmental expression pattern of this gene was assayed with qRT-PCR. The expression of TH gene in the 5th instar larva of H. armigera after treatment with 20E (20-hydroxyecdysone) (400 ng/individual) for different time and RNAi of ecdysone receptor gene (EcR) followed by 20E treatment (400 ng/individual) was analyzed by qRT-PCR, respectively. The TH acitivity in the fat body of H. armigera larvae after treatment with bio-stimulants bursicon (30 μg/mg tissue) and cAMP (200 ng/mg tissue) was determined by biochemical method. 【Results】 We cloned the full-length cDNA sequence of TH(GenBank accession no.: MF440319) from H. armigera, which is 2 270 bp in length with a 1 686 bp open reading frame encoding 561 amino acids. TH is ubiquitously expressed in all the examined developmental stages of H.armigera and relatively highly expressed in day-3 of egg stage, day-1 of the 2nd instar larval stage, 3rd-5th instar molting stages, prepupal stage and 1 day-old adult. In vitro treatment, the steroid hormone 20E (400 ng/individual) significantly up-regulated the transcription level of TH, but the 20E treatment after RNAi-mediated knockdown of 20E receptor gene EcR showed no obvious effect on the transcription of TH as compared with the control (injected with dsGFP only). Enzymatic activity assay demonstrated that both bursicon (30 μg/mg tissue) and cAMP (200 ng/mg tissue) led to significant increase of the TH activity. 【Conclusion】 20E is involved in the regulation of TH expression at the transcriptional level, and both bursicon and cAMP can promote the TH activity at the protein level.

Key words: Helicoverpa armigera; tyrosine hydroxylase, RNAi, 20-Hydroxyecdysone, bursicon