Cytochrome P450 genes compose a superfamily in insects, acting as the terminal oxidase in the monooxygenase system coupled with an electron donor such as NADPH-cytochrome P450 reductase (CPR). Recent studies have shown that CYP4G subfamily genes have a considerable role in cuticle formation and hydrocarbon synthesis in insects. However, CYP4Gs-related phenotypes and functions need to be further explored in beetles. In this study, CPR and two CYP4G genes, CYP4G7 and CYP4G14, were identified in Tribolium confusum. CPR, CYP4G7 and CYP4G14 were detected during different developmental stages and highly expressed at a late stage of pupae, with the levels of CYP4G14 much higher than those of CPR and CYP4G7 in T. confusum. Microinjection-based RNAi was applied to T. confusum and all three genes obtained over 50% reduce in expression levels in adults. RNAi of three genes performed in sixth-instar larvae resulted in higher than 60% mortalities in T. confusum during eclosion and cuticular pigmentation was inhibited in adults, leading to light coloration in adults. dsRNA injection in pupae led to defects in T. confusum eclosion and cuticular pigmentation. Abnormal adults had deformed epicuticle confirmed with scanning electron microscope and died within two weeks after eclosion. The results illustrated that CYP4G7 and CYP4G14, coupled with CPR, mediated cuticle formation and pigmentation in adults. The results provided new insights into the function of CYP4G subfamily genes in insects.

细胞色素 P450 基因在昆虫中构成一个超家族，作为单加氧酶系统中的末端氧化酶，与电子供体如 NADPH-细胞色素 P450 还原酶 (CPR) 结合。最近的研究表明，CYP4G 亚家族基因在昆虫角质层形成和碳氢化合物合成中具有相当大的作用。然而，需要在甲虫中进一步探索 CYP4Gs 相关的表型和功能。在这项研究中，在Tribolium confusum中鉴定了CPR和两个 CYP4G 基因，CYP4G7和CYP4G14。CPR、CYP4G7和CYP4G14在不同发育阶段被检测到，并在蛹后期高表达，其水平为在T. confusum中CYP4G14远高于CPR和CYP4G7。基于显微注射的 RNAi 应用于T. confusum，获得的所有三个基因在成人中的表达水平降低了 50% 以上。在六龄幼虫中进行的三个基因的 RNAi 导致T. confusum在羽化期间的死亡率高于 60% ，成虫的表皮色素沉着受到抑制，导致成虫颜色较浅。在蛹中注射 dsRNA 导致T. confusum羽化和表皮色素沉着缺陷。异常成虫经扫描电镜证实上表皮畸形，羽化后两周内死亡。结果表明CYP4G7和CYP4G14与 CPR 相结合，介导了成人的角质层形成和色素沉着。该结果为 CYP4G 亚家族基因在昆虫中的功能提供了新的见解。