β-Galactosidase is a critical exoglycosidase involved in the hydrolysis of lactose, the modification and degradation of glycoprotein in vivo. In this study, the β-galactosidase gene of silkworm (BmGal), whose cDNA comprises 11 exons and contains an intact ORF of 1,821 bp, was cloned. The protein sequence of BmGal showed high similarity with other known insect β-galactosidases. No activity of the BmGal expressed in Escherichia coli or Pichia pastoris was detected while it was successfully expressed with high enzyme activity in baculovirus expression system in silkworm, and the electrophoresis result revealed that the BmGal showed activity in oligomer mode. Enzyme activity assay showed that its optimum pH was 8.4 and its optimum temperature was 40 °C. What is more, we found that iron ions can stimulate the activity of the enzyme while cobalt, nickel, or lead ions can inhibit its activity significantly. Besides, the temporal–spatial transcription pattern of the BmGal mRNA level was analyzed, which showed that BmGal was transcribed at the highest level in the fifth larval instar but relatively low level in the pupal and adult stage, and the highest transcriptional level of BmGal was found in testis among all the tissues concerned.

中文翻译：

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家蚕新型β-半乳糖苷酶的克隆、表达及特性分析

β-半乳糖苷酶是一种关键的外切糖苷酶，参与乳糖的水解、体内糖蛋白的修饰和降解。本研究克隆了家蚕β-半乳糖苷酶基因（BmGal），该基因的cDNA由11个外显子组成，完整的ORF为1821 bp。BmGal的蛋白质序列与其他已知的昆虫β-半乳糖苷酶具有高度相似性。在大肠杆菌或毕赤酵母中表达的 BmGal 没有活性在家蚕杆状病毒表达系统中以高酶活性成功表达时检测到，电泳结果显示BmGal以寡聚模式显示活性。酶活性测定表明其最适pH为8.4，最适温度为40℃。更重要的是，我们发现铁离子可以刺激酶的活性，而钴、镍或铅离子可以显着抑制酶的活性。此外，分析了BmGal mRNA水平的时空转录模式，发现BmGal在幼虫5龄的转录水平最高，而在蛹和成虫阶段的转录水平相对较低，而BmGal的转录水平最高。在所有相关组织的睾丸中都发现了。