菌物研究2025，Vol.23Issue(4)：304-312,9.DOI:10.13341/j.jfr.2023.1685
荷叶离褶伞漆酶基因LdLac3克隆、原核表达及表达条件优化
Cloning,Prokaryotic Expression and Optimizing Expression Conditions of Laccase Gene LdLac3 in Lyphyllum decastes
摘要
Abstract
A laccase gene,LdLac3,was cloned from Lyophyllum decastes using the RT-PCR technique for investigation of its molecular function.Structural and physicochemical analysis of its encoded protein was conducted through the construction and transformation of an expression vector,pET-28a-LdLac3,into the prokaryotic cell Escherichia coli BL21.Our results indicated that the laccase gene LdLac3 had a sequence length of 1 623 bp,carrying a complete open reading frame(ORF)that encoded a number of 541 amino acids.The Lyophyllum decastes LAC3 protein was confirmed to have a molecular mass of 58 836.50 Da,an isoelectric point of 6.30,an instability index of 36.39 and a grand average of hydropathicity(GRAVY)of-0.178,indicating that the LDLAC3 was a kind of stable hydrophilic proteins.The constructed prokaryotic expression vector,pET-28a-LdLac3,was successfully found to express the recombinant protein in E.coli BL21 actively.Optimal expression activity of the recombinant protein in BL21 was investigated furthermore under the single-factor test conditions,including different shaking speeds,IPTG concentrations,induction times,induction temperatures and buffers using SDS-PAGE,which showed that the highest expression level of the recombinant protein LdLac3 was achieved at the induction temperature of 30℃,shaking speed of 200 r/min,induction time of 6 h,and IPTG concentration of 0.6 mmol/L.The above findings obtained at this study thus provide important research foundations and technical supports for future in-depth analysis of the laccase gene function and its application in industrial catalysis as well.关键词
漆酶/基因克隆/原核表达条件优化Key words
lactase/gene cloning/optimization of prokaryotic expression conditions分类
农业科技引用本文复制引用
王旺,梁倩倩,张瑞华,汪军成,牛鑫..荷叶离褶伞漆酶基因LdLac3克隆、原核表达及表达条件优化[J].菌物研究,2025,23(4):304-312,9.基金项目
甘肃省重点研发计划项目(23YFNG0006),张掖市重大技术攻关揭榜挂帅制项目(ZY2022JBGS07),甘肃省省级重点人才项目(2023RCXM34),国家自然科学基金地区基金项目(31860582),国家级大学生创新创业训练计划项目(S202110740028) （23YFNG0006）
