近暗散白蚁β-葡糖苷酶基因RpBg7的克隆及其在毕赤酵母中的表达  

Cloning of theβ-glucosidase gene RpBg 7 from Reticulitermes perilucifugus(Blattariae:Rhinotermitidae)and its expression in Pichia pastoris

作　　者：苏丽娟[1] 肖元玺 李琰[1] 伍志伟 赵鹏飞[1] 楚君鹏 魏纪珍 安世恒[2] 尹新明[2] 宋安东[1] SU Li-Juan;XIAO Yuan-Xi;LI Yan;WU Zhi-Wei;ZHAO Peng-Fei;CHU Jun-Peng;WEI Ji-Zhen;AN Shi-Heng;YIN Xin-Ming;SONG An-Dong(College of Life Sciences of Henan Agricultural University,Zhengzhou 450002,China;College of Plant Protection,Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南农业大学生命科学学院,郑州450002 [2]河南农业大学植物保护学院,郑州450002

出　　处：《昆虫学报》2020年第9期1039-1047,共9页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(31170350);河南省基础与前沿技术研究项目(142300410150);河南省高等学校重点科研项目(20A210004)。

摘　　要：【目的】白蚁是自然界中利用木质纤维素能力很强的生物,是纤维素酶的天然资源库。本研究旨在挖掘新来源的纤维素酶基因,为生物质能源的高效利用提供新的天然酶。【方法】根据前期蛋白质组测序的结果,利用PCR结合RACE克隆了近暗散白蚁Reticulitermes perilucifugusβ-葡糖苷酶7(β-glucosidase 7)基因RpBg7 cDNA全长序列;通过生物信息学软件分析了RpBg7的序列;用表达载体pPICZαA在毕赤酵母Pichia pastoris X-33中表达RpBg7蛋白,并用4-硝基苯基-β-d-吡喃葡萄糖苷(4-nitrophenylβ-d-glucopyranoside,4pNPG)为底物检测了表达的RpBg7蛋白的酶活性。【结果】获得了近暗散白蚁的一个内源性β-葡糖苷酶7基因RpBg7(GenBank登录号:MN944395),其开放阅读框长1485 bp,编码495个氨基酸残基。RpBg7蛋白预测分子量为57 kD,属于糖苷水解酶1(glycoside hydrolase 1,GH1)家族,具有保守的碱性氨基酸残基Glu187和Glu394。通过毕赤酵母表达系统成功表达RpBg7蛋白。酶活性分析结果表明,毕赤酵母胞外分泌蛋白粗酶液和胞内蛋白粗酶液中RpBg7酶活性分别为4.43和7.47 U/mL。【结论】克隆并利用毕赤酵母表达了近暗散白蚁的GH1家族的一个β-葡糖苷酶7基因RpBg7,为后期纤维素酶的改造和应用提供了条件。【Aim】Termites are the organisms with the high efficiency to use lignocellulose in nature,and are the natural resource reservoir of cellulases.This study aims to explore cellulase genes of new sources and to provide new natural enzymes for efficient utilization of biomass energy.【Methods】Based on the previous proteome analysis,the full-length cDNA sequence ofβ-glucosidase 7 gene(RpBg 7)was cloned from Reticulitermes perilucifugus by using RCR and RACE technology,and the RpBg7 sequence was analyzed by bioinformatic software.The RpBg7 protein was expressed with expression vector pPICZαA in Pichia pastoris X-33,and the enzyme activity of the expressed RpBg7 protein was assayed with 4-nitrophenylβ-d-glucopyranoside(4pNPG)as the substrate.【Results】We obtained an endogenousβ-glucosidase 7 gene,RpBg7(GenBank accession no:MN944395),from R.perilucifugus.Its open reading frame is 1485 bp in length,encoding 495 amino acids.The RpBg7 protein has the predicated molecular weight of 57 kD and belongs to glycoside hydrolase 1(GH1)family with conservative basic amino acid residues Glu187 and Glu394.The RpBg7 protein was successfully expressed in the P.pastoris expression system.The enzyme activity assay results showed that the enzyme activity of RpBg7 in the crude solutions of extracellular secretion proteins and intracellular proteins of P.pastoris was 4.43 and 7.47 U/mL,respectively.【Conclusion】Theβ-glucosidase 7 gene of GH1 in R.perilucifugus was cloned and expressed in P.pastoris,providing conditions for the modification and application of cellulases afterwards.

关 键 词：近暗散白蚁 Β-葡糖苷酶 纤维素酶 毕赤酵母 木质纤维素 

分 类 号：Q965.9[生物学—昆虫学]