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