暗黑鳃金龟幼虫羧酸酯酶基因的克隆及其在不同组织中的表达水平研究

Cloning and expression in different tissues of carboxylesterase gene from Holotrichia parallela larvae

通过对暗黑鳃金龟幼虫中肠cDNA文库的免疫筛选,得到一个编码羧酸酯酶的全长基因hc19。经NCBI Blast分析,该基因编码的氨基酸序列包含有Esterase/lipase superfamily的保守结构域,含羧酸酯酶B型丝氨酸活性位点FGGdpnkVtLaGySAG。序列比对表明,其与华北大黑鳃金龟有较高同源性。hc19在大肠杆菌中表达大约59kDa的目的蛋白条带。以α-醋酸萘酯溶液为底物与粗酶液混合,测其活性,平均酶活力为0.091mmol/100μL酶液。实时定量PCR表明羧酸酯酶基因hc19在中肠中表达量最高。

A full-length cDNA library of Holotrichia parallela was constructed with creator SMART eDNA construction Kit, and a carboxylesterase eDNA was obtained by immuno- screening the eDNA library with an anti-PM proteins polyelonal antiserum. NCBI Blast of the protein sequence revealed the highly conserved Esterase/lipase superfamily sites and the car- boxylesterases type-B serine active site FGGnpdsVtIsGqSAG. The deduced amino acid se- quence showed a high identity to the reported carboxylesterase from coleopteran insect Ho- lotrichia oblita. Western blot analysis showed that HC19 was successfully expressed in E. co- ll. Carboxylesterase activity assay in vitro showed that the recombinant HC19 could hydrolyze α-naphthyl acetate. Analysis of tissue-dependent expression revealed that the transcript is pri- marly expressed in the midgut by qRT-PCR approach, which provides a foundation for the further research of function of H. parallela carboxylesterase.