黄肢散白蚁β-糖苷酶基因的克隆及特性分析（英文）

Molecular cloning and characterization of a β-glycosidase gene from the termite Reticulitermes flaviceps( Isoptera: Rhinotermitidae)

【目的】黄肢散白蚁Reticulitermes flaviceps是我国代表性的食木白蚁之一,具有高效的纤维素酶系统。本研究旨在克隆黄肢散白蚁中纤维素酶基因并进行其特性分析,为白蚁纤维素酶资源的开发提供理论基础。【方法】采用反转录PCR(RT-PCR)、简并PCR以及c DNA末端快速扩增实验(RACE)法克隆黄肢散白蚁β-糖苷酶基因序列并进行生物信息学分析;利用大肠杆菌Escherichia coli原核表达该基因,表达出的蛋白质经纯化后进行纤维素酶活力测定。【结果】从黄肢散白蚁中克隆到一个β-糖苷酶基因GZRfbg1(Gen Bank登录号:KU886065)的全长c DNA序列,全长1 691bp,开放阅读框1 488 bp,编码448个氨基酸残基,预测分子量为56.45 k D,具有典型的糖基水解酶家族1(GHF1)的蛋白功能结构域。氨基酸序列多重比对显示,GZRfB G1与北美散白蚁Reticulitermes flavipes的唾液腺β-glucosidase氨基酸序列一致性为95%。GZRfB G1在大肠杆菌Rosetta 2(DE3)中进行体外表达,经纯化的GZRfB G1纤维素酶活力测定结果显示,其对水杨苷、羧甲基纤维素钠(CMC)以及纤维二糖的水解活性依次上升,能够在CMC-琼脂平板中产生清晰的水解圈。【结论】结果表明,来自黄肢散白蚁与北美散白蚁两个近缘白蚁的唾液腺β-糖苷酶在氨基酸序列及其水解特征方面亦具有高度保守性。该β-糖苷酶基因的研究丰富了白蚁纤维素酶开发及系统发生研究的理论基础。

[ Aim ] Reticulitermes flaviceps is one of the representative wood-feeding termite in our country, which has efficient cellulolitic system. This study aims to clone and characterize cellulase gene from R. flaviceps, so as to provide the theoretical basis for exploring cellulose resource of termites. [ Methods ] A β- glycosidase gene was cloned from R. flaviceps by using reverse-transcription polymerase chain reaction （ RT- PCR）, degenerate PCR and rapid amplification of cDNA end （RACE） methods, and its sequence was analyzed by bioinformatics tools. The corresponding protein was prokaryoticly expressed in Escherichia coli and the cellulase activity was measured after purification. [ Results ] The full-length cDNA sequence of β-glucosidase gone GZRfogl （GenBank accession no. ： KU886065 ） was cloned from R. flaviceps, with a length of 1 691 bp and an open reading frame of 1 488 bp, encoding 448 amino acid residues with the typical structure of glycosyl hydrolase family （GHF） 1 domain. The calculated protein molecular weight is 56.45 kD. Multiple alignment analysis revealed that the deduced amino acid sequence of GZRfBG1 shares 95% identity with the salivary gland 13-glucosidase of Reticulitermes flavipes. GZRfBG1 was heterologously expressed in E. coli Rosetta 2 （ DE3 ）. The purified tagged GZRfBG1 exhibited increased specific activity to salicine, carboxymethyl cellulose sodium （CMC）, and cellobiose. And the cellulolytic activity was also shown in a CMC containing agarose plate, in which clear digestion haloes were formed. [ Conclusion ] These results suggested that the salivary gland β- glucosidase from sibling species R. flaviceps and R. flavipes are very conservative in sequence and hydrolysis characteristics. This study of β-glucosidase gene can enrich the theoretical basis for the development of termite cellulases and phylogenetic study.