柞蚕蛹滞育相关的KK-42结合蛋白基因的功能分析

柞蚕的蛹滞育一直是柞蚕学乃至昆虫学领域的一个基础性理论问题，该问题的阐明在理论和应用上均有重要意义。KK-42结合蛋白是昆虫生长调节剂KK-42的结合蛋白,已发现其与天蚕的卵滞育有直接关系。前期研究中，我们分离了柞蚕的KK-42结合蛋白基因，进化分析发现KK-42 结合蛋白是一种新型的微卵黄蛋白，但其基因在4个发育阶段和各个组织中均有表达；蛋白序列含有潜在的脂肪酶活性功能域；已经明确其表达与柞蚕的蛹滞育也密切相关。本项目将深入研究该基因的生物学功能，包括：不同发育时期KK-42处理时该基因的应答情况，分析其与滞育激素基因等滞育相关基因及环境因素的相互作用；利用免疫细胞化学技术进行亚细胞定位；利用酶学方法对该蛋白的脂肪酶活性进行鉴定；利用RNAi技术进行该基因的功能分析。结果可以为理解柞蚕蛹滞育的分子机理提供新的线索，也为探明KK-42 的分子作用机制提供线索。

The Chinese oak silkworm, Antheraea pernyi, is a special economic insect distributed in more than ten Provinces in China. The mechanism of pupal diapause of this species is a key theorical problem to be clarified whithin the field of Sericology and Entomology. Imidazole derivative KK-42 is an insect growth regulator. KK-42 binding protein (KK-42BP) was first isolated from eggs of A. yamamai, and has been shown to be related to diapause in pharate first instar of A. yamamai. We have isolated the KK-42BP gene from A. pernyi (ApKK-42BP) that enters diapause as a pupa, supported by grant from the National Natural Science Foundation of China (No. 31072082). Our result suggests that KK-42BP is a new member of the minor yolk protein; however, its mRNA expression is present in all of the tissues tested, throughout four developmental stages, and in both female and male. Sequence analysis also show that KK-42BP has potential lipase motif.It has been evident that the KK-42BP gene is closely related to pupal diapause of A. pernyi. Based on these findings, we will further study the biological functions of KK-42BP in this project,as following: 1) Respones of the KK-42BP gene on treatment of KK-42 during four different developmental stages of A. pernyi; 2) Interaction between ApKK-42BP and known diapause-related genes, such as diapause hormone gene, in other insects and environmental factors; 3) Subcellular localization of ApKK-42BP by immunocytochemical technique; 4) Identification of the predicted lipase activity of ApKK-42BP by enzymological method after eukaryotic expression; 5) Function analysis of KK-42BP in A. pernyi by RNA inference. This project will provide a new clue to understand the molecular mechanism of pupal diapause in A. pernyi, and provide an evidence on molecular mechanism of actin of KK-42.