从富集液中发掘麻类脱胶果胶酶基因的技术  

Technique for Exploring Pectinase Gene of Bast Fiber Degumming from Enrichment Liquid

作　　者：段盛文[1] 刘正初[1] 郑科[1] 冯湘沅[1] 成莉凤[1] 郑霞[1] 

机构地区：[1]中国农业科学院麻类研究所,长沙410205

出　　处：《中国生物工程杂志》2014年第1期86-89,共4页China Biotechnology

基　　金：国家微生物资源平台专项经费(NIMR(2012)-01-03);国家"863"计划(2012AA022209D);湖南省自然科学基金(11JJ4021);国家麻类产业技术体系建设专项(CARS-19-E24)资助项目

摘　　要：为了突破传统可培养技术筛选麻类脱胶用果胶酶基因的不足,通过设计不同总DNA提取方法、样品富集方法以及优化PCR反应体系,建立起了一套适合于直接从富集液中发掘麻类脱胶用果胶酶基因的技术。结果表明,震荡培养脱胶时间比静止培养脱胶时间缩短51.5%;通过PowerSoilTMDNA Isolation Kit从第4次富集后的震荡发酵中能提取适合的总DNA;PCR最优反应体系为:总体积为25 ml,Mg2+浓度为2.0 mmol/L,dNTPs浓度为0.2 ng/μl,引物浓度为0.6μmol/L,DNA模板取2.5 ng/μl,Taq聚合酶量为0.8 U。获得的基因序列与已报道的果胶酶基因FJ538208序列高度相似,应用该技术成功地从麻类脱胶富集液中克隆到了果胶酶基因。To break through the deficiency of traditional culture technique in screening pectinase gene for bast fiber bio-degumming, different methods of total DNA extraction, sample enrichment, and optimization of PCR reaction system were designed, and a set of metagenomic technique that was suitable for excavation of pectinase gene for bast fiber degumming was established. The results showed that, degumming time of shake cultivation was shorter 51.5% than that of static cultivation to enrich microorganisms of ramie degumming; the total DNA for the study of metagenomic technique was extracted from the fourth fermentation by the PowerSoil？ DNA Isolation Kit; the PCR optimal reaction system in the total volume of 25 ml： the concentrations of Mg2＋ , dNTPs, primer, DNA template were 2. 0 mmol/L, 0. 2 ng/μl, 0. 6 μmol/1, 2. 5 ng/μL, and 0. 8 U respectively. The gene sequence obtained by the optimized technique was completely similar to the peetinase gene FJ538208 sequence reported.

关 键 词：麻类脱胶果胶酶基因 富集液 

分 类 号：Q815[生物学—生物工程]