苎麻脱胶果胶裂解酶基因的高效表达及序列分析  

High Level Expression of Pectate Lyase Gene from Microbiology for Ramie Degumming and Its Bioinformatics Analysis

作　　者：徐欢 段盛文[1] 冯湘沅[1] 郑科[1] 杨琦[1] 汪启明[2] 成莉凤[1] 彭源德[1] XU Huan;DUAN Shengwen;FENG Xiangyuan;ZHENG Ke;YANG Qi;WANG Qiming;CHENG Lifeng;PENG Yuande(Institute of Bast Fiber Crops,Chinese Academy of Agriculture Sciences,Changsha 410205,China;College of Bioscience and Biotechnology,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]中国农业科学院麻类研究所,湖南长沙410205 [2]湖南农业大学生物科学技术学院,湖南长沙410128

出　　处：《华北农学报》2021年第5期18-23,共6页Acta Agriculturae Boreali-Sinica

基　　金：中央级公益性科研院所基本科研业务费专项(1610242021002);湖南省自然科学基金(2019JJ40332,2019JJ50711);国家创新工程(ASTIP-IBFC08);国家现代农业产业技术体系建设专项(CARS-16-E22);湖南省重点领域研发计划(2019NK2071);长沙市科技计划项目(kq1901112)。

摘　　要：为使苎麻生物脱胶果胶裂解酶PelG403高效表达。将pelG403从重组质粒pEASY-Blunt E1-G403更换至质粒拷贝数更高的pET28a载体中,导入Escherichia coli BL21(DE3)后进行诱导表达。采用DNS法进行酶活力测定,通过SDS-PAGE和Western Blot分析检验PelG403表达效果,并对其序列进行分析鉴定。结果表明:pET28a-G403/BL21的果胶裂解酶活力为335.8 U/mL,较pEASY-Blunt E1-G403/BL21提高了3.05倍;工程菌pET28a-G403/BL21表达产物分子量比预测带His标签的融合蛋白分子量(43.6 ku)略小;PelG403含387个氨基酸,N末端前35个氨基酸为信号肽;理论pI值为7.64,有4个半胱氨酸,不稳定系数为27.42;其蛋白结构域含有典型的右手β-螺旋结构,属于Pec lyase C家族。因此,将pelG403从重组质粒pEASY-Blunt E1-G403更换至pET28a载体中,并导入E.coli BL21(DE3)进行诱导表达,是苎麻脱胶果胶裂解酶基因pelG403高效表达的方法和途径,同时对其序列进行分析鉴定,为果胶裂解酶的纯化、关键位点分析、分子改造及其脱胶功能特性研究奠定基础。In order to efficiently express the pectate lyase PelG403 from ramie biodegumming.This study replaced pel G403 from the recombinant plasmid pEASY-Blunt E1-G403 to the pET28a vector with a higher plasmid copy number,and then introduced Escherichia coli BL21(DE3)to induce expression.The enzyme activity was determined by DNS method,and the expression effect of PelG403 was tested by SDS-PAGE and Western Blot analysis,and the sequence was analyzed and identified.The results showed that the pectate lyase activity of pET28a-G403/BL21 was 335.8 U/mL,which was 3.05 times higher than that of pEASY-Blunt E1-G403/BL21;the molecular weight of the expressed product of pET28a-G403/BL21 was slightly smaller than that of the predicted fusion protein with His tag(43.6 ku);PelG403 contained 387 amino acids,and the first 35 amino acids at the N-terminal were signal peptides;the theoretical pI value was 7.64,4 cysteines,and the instability coefficient was 27.42;its protein domain contained a typical right-handedβ-helix structure and belonged to the Pec lyase C family.Therefore,replacing pelG403 from the recombinant plasmid pEASY-Blunt E1-G403 into the pET28a vector and introducing E.coli BL21(DE3)for inducible expression was a method and approach for high-efficiency expression of the ramie degummed pectate lyase gene pelG403,and its sequence was analyzed and identified.It laid a foundation for the purification of pectate lyase,key site analysis,molecular modification and the study of its degumming functional characteristics.

关 键 词：苎麻 生物脱胶 果胶裂解酶 原核表达 生物信息学 

分 类 号：Q78[生物学—分子生物学]