CRISPR/Cas9-mediated Tyrosine hydroxylase knockout resulting in larval lethality in Agrotis ipsilon
CRISPR/Cas9-mediated Tyrosine hydroxylase knockout resulting in larval lethality in Agrotis ipsilon
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CRISPR/Cas9介导的酪氨酸羟化酶敲除导致小地老虎幼虫死亡
DOI：
10.1111/1744-7917.12647
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发表时间：
2018
期刊：
影响因子：
4
通讯作者：
He Lin
中科院分区：
文献类型：
--
作者：
Yang Yang;Wang Yao Hui;Chen Xi En;Tian Di;Xu Xia;Li Kai;Huang Yong Ping;He Lin
酪氨酸羟化酶（Tyrosine hydroxylase， TH）参与昆虫黑色素和儿茶酚胺生物合成途径。TH作为催化酪氨酸转化为3,4‐二羟基苯丙氨酸的酶是该途径的第一步反应。虽然TH基因已被证实影响许多昆虫表皮的色素沉着和发育，但尚未有关于该基因在农业中的生理功能的报道。这里我们克隆了a的基因。ipsilon。半定量实时聚合酶链反应（PCR）分析显示aith在所有发育阶段均有表达。此外，它在头部和表皮的高表达表明它主要与色素沉积和昆虫发育有关。然后，我们使用聚类规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9系统靶向aithgene：在靶位点检测到缺失事件。与对照组相比，少数突变体出现蛋壳和胚胎变窄现象，能发育但不能孵化；其他孵化的胚胎在孵化后严重脱水，并在第一天内死亡。实时荧光定量PCR分析显示，该基因在突变体中表达下调。我们的工作表明，aith在新孵化的幼虫的生长发育中起着重要的作用；同时，探索a的控制策略将是一个有希望的目标。ipsilon。
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 theTHgene inAgrotis ipsilon. Here we cloned theTHgene fromA. ipsilon. Semi‐quantitative real‐time polymerase chain reaction (PCR) analysis showed thatAiTHwas 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 theAiTHgene: 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 thatTHwas down‐regulated inAiTHmutants. Here, our work demonstrated thatAiTHplays an important role in growth and development of newly hatched larvae; meanwhile, it would be a promising target to explore a control strategy forA. ipsilon.