柞蚕蛹滞育相关的KK-42结合蛋白基因的功能分析

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.

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。

KK-42是一种可以调节昆虫生长发育的咪唑类化合物，尤其是能解除天蚕和舞毒蛾的卵滞育。KK-42结合蛋白最早在天蚕中分离出来，且与其卵滞育解除直接相关。我们从以蛹滞育的柞蚕中也分离出该蛋白，其与天蚕KK-42结合蛋白的序列相似性达到95%。项目主要探讨了KK-42对柞蚕蛹滞育的影响和KK-42结合蛋白基因的生物学功能。（1）通过对3种蛹滞育研究的模式昆虫（柞蚕Antheraea pernyi、棉铃虫Helicoverpa zea和麻蝇Sarcophaga crassipalpis）的研究，发现KK-42的功能是延迟蛹滞育的解除过程，幼虫期处理则可显著提高蛹滞育的几率。（2）通过对柞蚕前胸腺（KK-42的靶标器官）的转录组分析，鉴定了柞蚕蜕皮激素合成途径相关基因以及促前胸腺激素信号途径相关基因，但没有检测到KK-42结合蛋白基因的表达，说明KK-42对其靶器官前胸腺的影响并不是通过KK-42结合蛋白的途径。（3）成功进行了KK-42结合蛋白的原核表达，产物纯化后进行了脂肪酶活性的检测，结果表明KK-42结合蛋白虽然含有脂肪酶功能域，但并不具有脂肪酶的活性。（4）成功制备了KK-42结合蛋白的抗体；利用qRT-PCR和Northern blot分析，表明KK-42结合蛋白的最终去向是柞蚕卵。该蛋白在脂肪合成，血液运输，最后运送到卵，在胚胎发育时逐渐消耗。（5）KK-42结合蛋白在进化上属于minor yolk protein家族成员，利用qRT-PCR比较了其与卵黄原蛋白和微卵黄原蛋白的表达模式，结果表明这三种卵黄蛋白基因展现完全不同的表达模式，KK-42结合蛋白的表达在注射20-E后的第10天才可以检测到，微卵黄原蛋白在第4天即可检测到，而卵黄原蛋白在整个滞育期和后滞育期均可检测出来。（6）结果表明，KK-42结合蛋白在柞蚕的生殖发育中起着重要作用。我们的结果也进一步证实了KK-42与蜕皮激素合成代谢途径的关联，并发现了KK-42与滞育激素信号途径的关联，为下一步的研究提供了新的线索。