Guangdong Provincial Key Lab of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, 55 W. Zhongshan Ave., Guangzhou 510631, China.
Insect Biochem Mol Biol. 2013 Sep;43(9):794-808. doi: 10.1016/j.ibmb.2013.06.001. Epub 2013 Jun 22.
Larval cuticle is degraded and replaced by the pupal counterpart during larval-pupal metamorphosis in the holometabolous insects. In addition to the extrinsic transformation, the epidermis goes through significant changes at molecular levels. To elucidate the intrinsic mechanism of epidermal metamorphosis, the dynamics of chitin content in the cuticle was examined in an important agricultural lepidopteran, the common cutworm, and the transcriptome was analyzed using Illumina sequencing technology. Gene expression profiles during the metamorphosis were further studied by both the digital gene expression (DGE) system and real-time quantitative PCR. The results showed that the chitin content decreased in prepupae and then increased in pupae. A total of 58 million sequencing reads were obtained and assembled into 70,346 unigenes. Over 9000 unigenes were identified to express differentially during the transformation process. As compared with the 6th instar feeding larvae, the most significant changes took place in the proteasome and metabolic pathways in prepupae and pupae, respectively. The cytochrome P450s, VHDLs, chitinase, serine protease and genes involved in sex pheromone biosynthesis changed their mRNA levels remarkably. Three chitinolytic enzymes (chitinase, β-N-acetylglucosaminidase and chitin deacetylase) showed distinct mRNA expression patterns, the former two enzymes revealed the highest expression in prepupae, however the latter one showed its climax mRNA level in pupae. The gene expression patterns suggest that chitinase and β-N-acetylglucosaminidase may be responsible for the degradation of larval cuticles, whereas chitin deacetylase may help to degrade the pupal counterparts. Gene expression dynamics also implied that the chitin of pupal cuticle might be formed by recycling of the degraded chitin of larval cuticle rather than through de novo synthesis. The 20E-induced nuclear receptors seem to be important factors regulating chitin metabolic enzymes during the cuticle remodeling. Our data provide a comprehensive resource for exploring the molecular mechanism of epidermal metamorphosis in insects.
在完全变态的昆虫中，幼虫表皮在幼虫-蛹变态期间被幼虫表皮的对应物降解和取代。除了外在的转化，表皮在分子水平上也经历了显著的变化。为了阐明表皮变态的内在机制，我们在重要的农业鳞翅目昆虫——小菜蛾中检查了表皮的几丁质含量的动态，并利用 Illumina 测序技术分析了转录组。通过数字基因表达（DGE）系统和实时定量 PCR 进一步研究了基因表达谱在变态过程中的变化。结果表明，几丁质含量在预蛹期减少，然后在蛹期增加。共获得 5800 万条测序reads，并组装成 70346 个单体。在转化过程中，有超过 9000 个基因被鉴定为差异表达。与第 6 龄期取食幼虫相比，在预蛹和蛹中，分别有最显著的变化发生在蛋白酶体和代谢途径中。细胞色素 P450s、VHDLs、几丁质酶、丝氨酸蛋白酶和参与性信息素生物合成的基因的 mRNA 水平显著改变。三种几丁质酶（几丁质酶、β-N-乙酰氨基葡萄糖苷酶和几丁质脱乙酰酶）表现出明显不同的 mRNA 表达模式，前两种酶在预蛹期表达最高，而后者在蛹期表现出最高的 mRNA 水平。基因表达模式表明，几丁质酶和β-N-乙酰氨基葡萄糖苷酶可能负责幼虫表皮的降解，而几丁质脱乙酰酶可能有助于降解蛹的对应物。基因表达动力学也暗示，蛹表皮的几丁质可能是通过降解的幼虫表皮的几丁质的再循环而不是从头合成形成的。20E 诱导的核受体似乎是调节表皮重塑过程中几丁质代谢酶的重要因素。我们的数据为探索昆虫表皮变态的分子机制提供了一个全面的资源。
