家蚕滞育性卵中NAD+降解增强导致呼吸耗氧量下降的分子机制

As other insects, the decline of oxygen consumption is found in the silkworm, Bombyx mori, during diapause initiation. Recently, it has been confirmed that the catabolite of oxidized nicotinamide adenine dinucleotide (NAD+) participate in the regulation of metabolism, signal transduction and expression of gene. In previous studies, we found that the enhancement of the NAD+ catabolism resulted in the reduction of NAD level (including NAD+ and its reduced form, NADH) in Bombyx diapausing eggs before diapause initiation. We hypthesize that the catabolite of NAD+ may directly or indirectly inhibit the activities of the enzymes related to the pathway of glycolysis (EMP), tricarboxylic acid cycle (TCA) and the respiratory electron transport chain (ETC), and further result in the decline of oxygen consumption. In this study, first, the catabolism of NAD+, EMP, TCA and ETC are compared among diapausing eggs, constantly hydrochloric acid-treated diapausing eggs and nondiapause eggs to classify the sorts of the catabolite and the key enzymes; second, the genes coding the key enzymes are cloned; third, expression of above genes, covalent modification, allosteric regulation, and feedback inhibition are respectively determined to investigate whether the activities of the key enzymes are regulated at above levels, and finally the molecular mechanisms underlying on the decline of oxygen consumption caused by the catabolism of NAD+ in Bombyx diapausing eggs. Moreover, the results obtained in this work may provide the clues to investigate the molecular mechanisms underlying on the decline of oxygen consumption in the other insects during diapause initiation.

与其它昆虫一样，滞育发动时家蚕呼吸耗氧量显著下降，但是其分子机制还不清楚。近年来大量研究证实，氧化型烟酰胺腺嘌呤二核苷酸（NAD+）降解产物参与代谢、信号传导和基因表达等调控。此前我们发现滞育发动前NAD含量显著下降，表明NAD+降解加强。我们认为，NAD+降解产物通过直接或间接抑制糖酵解-三羧酸循环和呼吸电子传递链酶活性，导致滞育发动时呼吸耗氧量下降。本项目拟通过比较家蚕滞育性卵、即时浸酸卵和非滞育性卵中NAD+降解、糖酵解、三羧酸循环和呼吸电子传递链，确定家蚕滞育性卵中NAD+降解产物和参与上述代谢调控的关键酶种类；克隆关键酶基因，调查关键酶活性调控是否发生在基因表达、蛋白质共价修饰、变构调节和反馈抑制等水平上；最终解明家蚕滞育性卵中NAD+降解增强导致呼吸耗氧量下降的分子机制。此外，还可以为探讨其它昆虫滞育发动时呼吸耗氧量下降的分子机制提供线索。

利用分光光度法、液相色谱法和转录组测序测定了滞育发动阶段二化性家蚕滞育性卵和即时浸酸阻止滞育发动卵中生物氧化特性和相关基因转录水平的变化。结果发现，糖酵解途径、三羧酸循环和呼吸电子传递链的限速物质和限速酶分别为丙酮酸与3磷酸甘油激酶、NADH与柠檬酸裂解酶和ATP与ATP合酶，其中ATP和ATP合酶是整个生物氧化的限速物质和限速酶；与呼吸耗氧量变化趋势一致的指标包括丙酮酸、NADH和ATP含量，NADH/NAD+比值，丙酮酸激酶、丙酮酸脱氢酶、柠檬酸合酶、琥珀酸脱氢酶、呼吸电子传递链复合体二和ATP合酶；与滞育发动相关的先导指标为丙酮酸含量；NAD+降解酶Sirtuins 7，生物氧化中磷酸烯醇丙酮酸羧激酶和乳酸脱氢酶等编码基因转录水平发生了显著变化。上述结果表明，家蚕滞育性卵在滞育发动阶段呼吸耗氧量的变化主要与丙酮酸含量变化有关，参与其NAD+降解代谢的主要是Sirtuins 7。