家蚕滞育卵氧化还原态调控机制的研究

Redox status plays an important role in the regulation of cellular division, differentiation and apoptosis, which changes are closely related with the metabolisms of H2O2, GSH, NAD and NADP. In previous studies, we found that redox status in diapause eggs of the silkworm, Bombyx mori, shifts to the reducing conditions at the initiation phase but the pro-oxidant conditions at the termination phase, respectively. To classify regulatory mechanisms of redox status, we are first to measure above metabolisms to find the key enzymes regulating redox status in diapause eggs of the silkworm using the method of HPLC, then clone the genes encoding the key enzymes, and finally investigate the relationship between changes in the key enzymes and levels of gene expression, covalent modification of protein phosphorylation and allosteric modification using the methods of qRT-PCR, western blot, protein phosphorylation and enzymology analysis. As the silkworm is the type organism of diapause, this study has important scientific meaning on the further understanding of the molecular mechanisms of diapause regulation in the silkworm and other insects.Moreover, as stress resistance of insects is significantly enhanced at diapause status, which is also closely related with above metabolisms,this study may provide indices of physiology and biochemistry.

氧化还原态在细胞分裂、分化和凋亡中具有重要调控作用，其变化与H2O2、GSH、NAD和NADP代谢密切相关。此前我们发现，家蚕滞育卵在滞育发动阶段向还原态转移，相反在滞育解除阶段向氧化态转移。为了弄清家蚕滞育卵氧化还原态的调控机制，我们利用HPLC方法测定不同滞育阶段家蚕滞育卵H2O2、GSH、NAD和NADP代谢，以确定参与其氧化还原态调控的关键酶种类；利用qRT-PCR、Western blot和蛋白质磷酸化检测技术以及酶学性质分析，以调查不同滞育阶段参与家蚕滞育卵氧化还原态调控的关键酶活性变化与其基因表达、磷酸化共价修饰和变构调节之间的关系。家蚕是昆虫滞育研究的模式物种，本研究对于深入理解家蚕乃至其它昆虫滞育调控的分子机制，有着重要的科学意义。此外，滞育显著提高昆虫抗逆性，上述代谢与也抗逆性密切相关，本研究还可以为家蚕抗逆育种提供生理生化指标。

此前我们发现，家蚕滞育卵在滞育发动阶段向还原态转移，相反在滞育解除阶段向氧化态转移。在本研究中，我们进一步测定了家蚕滞育卵NADP代谢及其调控关键酶基因表达的变化。在滞育发动阶段NAD激酶（NADK）活性滞育性卵下降并且显著低于非滞育性卵，导致滞育性卵NADP含量下降并且显著低于非滞育卵；6-磷酸葡萄糖脱氢酶（G6PDH）和异柠檬酸脱氢酶（ICDHc）活性滞育性卵显著低于于非滞育性卵；导致滞育性卵NADPH/NADP+比值显著升高是由于其NADK活性低。在滞育解除阶段，5 ℃处理50 d以上滞育卵NADK活性升高并且显著高于25 ℃处理的滞育卵，导致5 ℃处理NADP含量增加并且显著高于同期25 ℃处理的滞育卵；5 ℃处理30 d以上滞育卵G6PDH活性升高并且显著高于25 ℃处理的滞育卵，但是两种处理的ICDHc活性没有显著差异，高G6PDH活性导致5 ℃处理70~90 d滞育卵的NADPH/NADP+比值显著升高。克隆了家蚕nadk（NAD kinase）和烟酰胺核苷酸转氢酶tnn（nicotinamide nucleotide transhydrogenase）基因。利用Real time PCR技术进行了nadk和tnn转录水平分析。滞育发动阶段滞育性卵nadk mRNA水平缓慢下降，但是显著高于同期非滞育性卵，其变化与NADK活性变化不一致；滞育解除阶段5 ℃处理的滞育卵普遍高于25 ℃处理，其变化与NADK活性变化大体一致。tnn mRNA水平滞育发动早期（产下后24~36 h）滞育性卵显著高于非滞育性卵，后期（产下后48~72 h）滞育性卵低于非滞育性卵；滞育解除阶段5 ℃处理的滞育卵普遍高于25 ℃处理的滞育卵。利用Western blot技术进行了nnt翻译水平分析。nnt蛋白质相对含量在滞育发动阶段滞育性卵普遍高于非滞育性卵，相反，5 ℃处理的滞育卵普遍低于25 ℃处理的滞育卵。上述结果表明，家蚕滞育卵中NADP代谢与细胞氧化还原态调控密切相关。