Laboratory of Sericulture & Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Insect Biochem Mol Biol. 2011 Jun;41(6):378-87. doi: 10.1016/j.ibmb.2011.02.007. Epub 2011 Mar 4.
We previously cloned a cDNA for sorbitol dehydrogenase (SDH1) from Bombyx mori. In the present study we cloned two additional cDNAs encoding SDHs (designated as SDH2a and SDH2b). The amino acid sequences of SDH2ab were almost the same and had higher similarity to the SDHs of other organisms than to B. mori SDH1. The SDH2ab and SDH1genes were located in tandem within about 40 kbp on chromosome 21. SDH2ab mRNAs increased after exposing diapause eggs to 5 °C for 40 days, beginning at 2 days post-oviposition, to break diapause. However, they were at very low levels in diapausing eggs incubated at 25 °C continuously from oviposition. These changes in expression pattern of SDH2ab mRNA were almost the same as that of SDH1 mRNA. To understand whether SDH1 and SDH2 were responsible for the SDH activity seen in diapause eggs exposed to 5 °C for more than 60 days, we expressed a His-tagged SDH2a fusion protein in Escherichia coli and examined its enzymatic parameters. The maximum activity of SDH2a observed at pH 8.4∼9.0, and the Km value for sorbitol was 12.6 mM, similar to the kinetic properties of other SDHs. Due to the significantly higher similarity between SDH2a and b, they were thought to have similar kinetic properties. Therefore, we purified SDH from B. mori diapause-terminated eggs exposed to 5 °C for 300 days which showed higher SDH activity using two-step affinity chromatography. The highly purified SDH showed a higher Km value (125 mM) for sorbitol, being similar to the value (136 mM) determined previously from Eadie-Hofstee plots using egg crude extract as an enzyme source; additionally, the plots showed one slope indicating one Km value. Moreover, in silico analysis indicated that no SDH genes other than SDH1 and 2ab are present in B. mori genomic DNA. These results suggest that SDH1 activity may be responsible for the majority of the increased SDH activity seen in diapause eggs after acclimation to 5 °C rather than SDH2ab. Further, the relative sequence divergence among these genes is consistent with the idea/hypothesis that the original SDH gene was first duplicated into SDH1 and SDH2, and then SDH2 was duplicated into the SDH2a and SDH2b genes.
我们之前已经从家蚕中克隆了山梨醇脱氢酶（SDH1）的 cDNA。在本研究中，我们克隆了另外两个编码 SDH 的 cDNA（分别命名为 SDH2a 和 SDH2b）。SDH2ab 的氨基酸序列几乎相同，与其他生物体的 SDH 比与家蚕 SDH1 的相似性更高。SDH2ab 和 SDH1 基因位于染色体 21 上约 40 kbp 的串联位置。在 5°C 下暴露 40 天后，从产卵后第 2 天开始，SDH2ab mRNA 增加，打破滞育。然而，在 25°C 下连续孵化的滞育卵中，它们的水平非常低。SDH2ab mRNA 的这种表达模式的变化与 SDH1 mRNA 的变化几乎相同。为了了解 SDH1 和 SDH2 是否负责在 5°C 下暴露超过 60 天的滞育卵中的 SDH 活性，我们在大肠杆菌中表达了带有 His 标签的 SDH2a 融合蛋白，并检查了其酶学参数。在 pH 8.4∼9.0 时观察到 SDH2a 的最大活性，山梨醇的 Km 值为 12.6 mM，与其他 SDH 的动力学特性相似。由于 SDH2a 和 b 之间具有显著更高的相似性，因此它们被认为具有相似的动力学特性。因此，我们使用两步亲和层析法从在 5°C 下暴露 300 天的已终止滞育的家蚕中纯化 SDH，该 SDH 显示出更高的 SDH 活性。高度纯化的 SDH 对山梨醇的 Km 值（125 mM）较高，与以前使用卵粗提物作为酶源从 Eadie-Hofstee 图确定的值（136 mM）相似；此外，该图显示一条斜率表明一个 Km 值。此外，计算机分析表明，家蚕基因组 DNA 中不存在除 SDH1 和 2ab 之外的其他 SDH 基因。这些结果表明，SDH1 活性可能是适应 5°C 后滞育卵中 SDH 活性增加的主要原因，而不是 SDH2ab。此外，这些基因之间的相对序列差异与最初的 SDH 基因首先复制成 SDH1 和 SDH2，然后 SDH2 复制成 SDH2a 和 SDH2b 基因的想法/假设一致。
