家蚕孵化酶基因的表达调控与功能鉴定的研究

Hatching process is of great significance to the development of the individual metazoan animals,while molecular mechanism of silkworm egg hatching process is unclear. Recently, our group has cloned two hatching enzyme genes from Bombyx mori (BmHE I and BmHE II) and deduced their possible biological function. Based on the achieved results, the current program, "Functional identification and transcription regulation analysis of hatching-enzyme genes from the silkworm, Bombyx mori", are proposing. This proposal includes the following research contents. The transcription and expression locations of BmHE I and BmHE II in the embryo, larvae,pupa and moth stage will be determined by in situ hybridization and the immunohistochemisty methods; The BmHE I and BmHE II will be expressed in foreign expression system, and its enzymatic characters will be investigated.Thence by using the intact silkworm eggshells as substrates, the degradation activity of BmHEs will be investigated in vitro; Moreover, biological function of BmHE I and BmHE II, such as hatching, digestion and spermatogenesis, will be investigated in vivo through the RNAi technology. To get the information on BmHE I and BmHE II transcription regulation, the promoter region of BmHE I and BmHE II will be cloned and analyzed to get the candidate cis-elments, and the transcription factors will be identified by the methods of the gel retardation assays. Based upon the above research results, the molecular mechanism of silkworm eggs hatching process will be elucidated initially, which will be presented the practical theory to increase the hatchability and quality of silkworm eggs, but also the clues to control the other Lepidoptera agricultural pests efficiently.

孵化过程对后生动物个体发育具有重要意义，而鳞翅目重要模式生物家蚕卵孵化行为的分子机理尚未明了。在已克隆了2个家蚕卵孵化酶基因（BmHE I和BmHE II）基础上，本项目拟进一步开展BmHE I和BmHE II功能鉴定与转录调控研究。采用原位杂交法和免疫组化法确定BmHE I和BmHE II在胚胎、幼虫、蛹期和蛾期组织中的细胞定位；体外表达BmHE I和BmHE II，调查其酶学特征及对家蚕卵壳的作用；通过RNAi方法个体水平上进行功能验证，明确BmHE I和BmHE II在孵化、消化及精子发育等方面的生理功能。克隆分析BmHE I和BmHE II启动子序列，获得候选的顺式作用元件；利用蛋白质凝胶电泳阻滞法确定其结合的转录因子，初步探明其转录表达调控途径。研究结果将为阐明家蚕卵孵化的分子机理奠定基础，为提高蚕卵的孵化率和蚕种质量提供理论依据，同时为开发鳞翅目农林害虫防治新途径提供借鉴。

孵化过程对后生动物个体发育具有重要意义，而鳞翅目重要模式生物家蚕卵孵化行为的分子机理尚未明确。孵化酶是孵化过程中起关键作用的一类蛋白酶，本课题组在已克隆了 2 个家蚕卵孵化酶基因（BmHE I 和 BmHE II）基础上，本项目进一步开展 BmHE I 和 BmHE II 功能鉴定与转录调控研究。RT-PCR和RNAi实验结果显示两者都参与了蚕卵孵化过程；RT-PCR和原位杂交实验结果显示BmHE I在幼虫期中肠组织细胞内特异表达，表达时相与取食量呈现正相关；而BmHE II在幼虫五龄期、蛹期和蛾期的精巢细胞内中特异表达，表明其参与了精子发育、成熟的过程；体外表达系统表达两个孵化酶蛋白，进行了酶学特性分析，酶切活性位点一致，分别为Tyr,Leu和Glu，孵化酶活性抑制实验表明5 5'-二甲基-2,2-联吡啶(5,5'-dimethyl-2,2'-bipyridyl) 抑制效果最显著，呈现浓度依赖效应，20mM可以抑制孵化酶活性的92%，添食实验表明显著抑制家蚕消化吸收。克隆了1.5kb两个BmHE I 和 BmHE II启动子，酵母单杂交系统和EMSA法筛选、验证了Oct-1、CRE-BP1、TBP、HNF-4alpha1转录因子参与孵化酶基因组织时空特异性转录调控。这为阐明家蚕孵化酶基因转录调控网络途径提供实验基础,为提高蚕卵的孵化率和蚕种质量提供理论依据，同时为开发鳞翅目农林害虫防治新途径提供借鉴。