School of Life Science, East China Normal University, Shanghai, China.
Hubei Key Laboratory of Insect Resource Application and Sustainable Pest Control, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China.
Insect Sci. 2018 Dec;25(6):1017-1024. doi: 10.1111/1744-7917.12647.
Tyrosine hydroxylase (TH) is involved in insect melanin and the catecholamine biosynthesis pathway. TH as an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine is the first step reaction in the pathway. Although TH has been proven to affect the pigmentation of the epidermis and development in many insects, there is no report about physiological function of the TH gene in Agrotis ipsilon. Here we cloned the TH gene from A. ipsilon. Semi-quantitative real-time polymerase chain reaction (PCR) analysis showed that AiTH was expressed at all development stages. Moreover, its high expression levels in the head and epidermis suggest that it is mainly related to pigment deposition and insect development. Then, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system to target the AiTH gene: deletion events were detected at the target sites. Compared with the control group, a few mutants with the phenomenon of narrowing in the egg shell and embryos can develop but cannot hatch; the other hatched embryos were seriously dehydrated after hatching and died within the first day. Quantitative real-time PCR analysis revealed that TH was down-regulated in AiTH mutants. Here, our work demonstrated that AiTH plays an important role in growth and development of newly hatched larvae; meanwhile, it would be a promising target to explore a control strategy for A. ipsilon.
酪氨酸羟化酶（TH）参与昆虫黑色素和儿茶酚胺生物合成途径。TH 作为一种酶，可催化酪氨酸转化为 3,4-二羟苯丙氨酸，是该途径中的第一步反应。尽管 TH 已被证明会影响许多昆虫的表皮色素沉着和发育，但关于 TH 基因在斜纹夜蛾中的生理功能尚无报道。本文从斜纹夜蛾中克隆了 TH 基因。半定量实时聚合酶链反应（PCR）分析表明，AiTH 在所有发育阶段都有表达。此外，其在头部和表皮中的高表达水平表明它主要与色素沉积和昆虫发育有关。然后，我们使用成簇规律间隔短回文重复（CRISPR）/CRISPR 相关蛋白 9 系统靶向 AiTH 基因：在靶位点检测到缺失事件。与对照组相比，少数具有卵壳和胚胎变窄现象的突变体可以发育但不能孵化；其他孵化的胚胎孵化后严重脱水，并在第一天内死亡。定量实时 PCR 分析显示 AiTH 在 AiTH 突变体中下调。本文工作表明 AiTH 在新孵化幼虫的生长和发育中发挥重要作用；同时，它可能是探索斜纹夜蛾控制策略的有前途的靶标。
