State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China.
Insect Biochem Mol Biol. 2022 Jan;140:103701. doi: 10.1016/j.ibmb.2021.103701. Epub 2021 Dec 8.
Insect cuticular hydrocarbons (CHCs) have dual functions as physical barrier and chemical signals. The last step of CHC biosynthesis is known to be catalyzed by cytochrome P450 CYP4G in a number of insects. Until recently, studies on CYP4Gs in the context of functional evolution are rare. In this study, we analyzed sequence similarity and temporal-spatial expression patterns of the five CYP4G genes in the cotton bollworm Helicoverpa armigera, an important agricultural pest and also typical representative of lepidopteran insects. Moreover, the CRISPR/Cas9-induced knockout was used to clarify the roles of the five CYP4Gs in CHC biosynthesis. Temporal-spatial expression patterns revealed that CYP4G8 was highly expressed at all developmental stages and in most tissues examined. Larvae with CYP4G8 knocked out could not produce methyl-branched CHCs and failed to pupate, while larvae with the other four CYP4G genes knocked out (4G1-type-KO) showed no significant changes in their CHC profiles, weight gain and survival. Comparative transcriptomics revealed that knocking out CYP4G8 affected the global gene expression in larvae, especially down-regulated the expression of genes in the fatty acid biosynthetic pathway, while no significant change in 4G1-type-KO transcriptome was observed. These findings indicate that the five members of the CYP4G subfamily have undergone functional divergence: CYP4G8 maintains the essential function in CHC biosynthesis, while the function of the other four CYP4G genes remains unclear. Intriguingly, CYP4G8 has evolved to be a P450 enzyme responsible for the synthesis of larval methyl-branched hydrocarbons. The observation that CYP4G8 knockout is lethal strongly suggest that CYP4G8 may serve as a candidate target for the development of insecticidal agents for the control of cotton bollworms.
昆虫表皮碳氢化合物 (CHC) 具有物理屏障和化学信号的双重功能。在许多昆虫中，CHC 生物合成的最后一步被认为是由细胞色素 P450 CYP4G 催化的。直到最近，关于 CYP4G 在功能进化方面的研究还很少。在这项研究中，我们分析了棉铃虫 Helicoverpa armigera 中五个 CYP4G 基因的序列相似性和时空表达模式，棉铃虫是一种重要的农业害虫，也是鳞翅目昆虫的典型代表。此外，还使用 CRISPR/Cas9 诱导敲除来阐明这五个 CYP4G 在 CHC 生物合成中的作用。时空表达模式表明，CYP4G8 在所有发育阶段和大多数检查的组织中都高度表达。敲除 CYP4G8 的幼虫不能产生甲基支链 CHC，无法化蛹，而敲除其他四个 CYP4G 基因（4G1 型-KO）的幼虫在 CHC 谱、体重增加和存活率方面没有明显变化。比较转录组学表明，敲除 CYP4G8 会影响幼虫的全局基因表达，特别是下调脂肪酸生物合成途径中的基因表达，而 4G1 型-KO 转录组则没有明显变化。这些发现表明 CYP4G 亚家族的五个成员已经发生了功能分化：CYP4G8 维持了 CHC 生物合成的基本功能，而其他四个 CYP4G 基因的功能仍不清楚。有趣的是，CYP4G8 已经进化为一种负责合成幼虫甲基支链烃的 P450 酶。CYP4G8 敲除致死的观察结果强烈表明，CYP4G8 可能成为开发用于控制棉铃虫的杀虫剂的候选靶标。
