家蚕新突变体2龄不眠蚕基因的定位克隆及其分子机制

The ecdysone, moulting hormone,of insects plays important roles in their exuviation and metamorphism.Its titer varies with the rhythm of life activity of insects. Many genes involve in the process of ecdysone synthesis. Mmutants of these corresponding genes have provided excellent models for study of the function of genes in the passway of ecdysone synthesis. We have found a novel moultinism mutant silkworm (Bombyx mori) and termed as nm2 mutant,which can not exuviate in the 2nd moult to the 3rd instar. The nm2 mutant is dominated by a pair of recessive genes and its homezygote is lethal. But feeding the mutant larvae with 20-hydroxyecdysone(20E) can rescure them from dying and exuviate to the next instar.We therefore deduced that nm2 mutant associates with the low ecdysone titer in the larval hemolymph. In order to investigate the molecular machanism of nm2 mutant, we planned to determine the position of nm2 gene by posotional cloning approach,and analysize gene structure and its expression profile. Then biochemically determine the functional period of nm2 gene in the process of ecdysone synthesis. And validate the function of nm2 gene by using GAL4/USA system to interfere the gene for elucidating its molecular machanism. The discovery of nm2 mutant has enriched the silkworm resource and established a novel natural model for study of ecdysone synthesis in insects. And our results will put a solid foundation for elucidating the machanism of development and metamorphism of insects,and study of nm2 gene will show a potential value in pest control.

昆虫蜕皮激素在昆虫的蜕皮和变态中起着重要作用，其滴度随着生命活动节律发生动态变化。许多基因参与蜕皮激素的合成，相应的突变体是证实这些基因在蜕皮激素合成途径中所扮演角色极佳模型。我们发现了一种家蚕新突变体-2龄不眠蚕（nm2），该突变体不能入眠蜕皮进入3龄，受1个隐性基因控制，纯合致死，添食蜕皮激素能拯救蜕皮进入下一龄期。因此，推测该突变体的发生与蜕皮激素滴度过低有关。为此，本项目拟采用图位克隆技术对nm2基因进行定位，分析该基因的结构特点和表达特征；用生化处理方法确定nm2基因在蜕皮激素合成途径作用时期；利用GAL4/UAS转基因技术干涉目标基因，验证该基因的功能。从分子水平揭示2龄不眠蚕突变体产生的本质。该突变体的发现丰富了家蚕品种资源，为完善家蚕蜕皮激素合成途径研究提供良好的模型，为阐明昆虫发育变态机制奠定基础。同时，研究利用该基因对害虫防治新技术开发具有潜在应用价值。

家蚕是一种完全变态昆虫，其幼虫发育过程中需要经过3-4次蜕皮，蜕皮激素和保幼激素起着调节作用。二龄不眠蚕突变体nm2在二龄期不能蜕皮而导致死亡。本课题以nm2为研究对象，采用图位克隆方法研究了突变基因的结构和突变体形成的分子机制，以进一步揭示家蚕蜕皮的本质。经定位克隆分析其候选基因为BMgn002602，即富含甘氨酸的表皮蛋白10（BmCPG10）基因，其ORF在其功能区缺失了217bp，RNAi实验证明BmCPG10基因是产生突变体的关键基因。经ELISA方法检测，与正常对照相比该突变体的蜕皮激素水平低下，用蜕皮激素合成的路径中的原材料胆固醇、中间产物7-脱氢胆固醇和终产物20E添食能回复突变体，qRT-PCR显示蜕皮激素的滴度与突变基因表达有着负相关关系。DEG测序结果表明部分编码表皮蛋白的基因、蜕皮激素生物合成通路上的基因及蜕皮激素信号通路上的核受体基因等的表达，在突变体和对照之间具有显著差异。综述实验结果，我们推测BmCPG10和蜕皮激素的相互影响关系， BmCPG10突变能够影响植物甾醇向胆固醇的转化，导致蜕皮激素滴度低下，同时，BmCPG10又是蜕皮激素信号通路上的效应物。本课题研究揭示了蜕皮激素在家蚕生长发育中的重要性及BmCPG10的新功能。