基于毛细管电泳的TILLING实验技术的建立  

Establishment of TILLING experimental technique based on capillary electrophoresis

作　　者：付强 邹颉 张吉顺 王轶 任学良 

机构地区：[1]贵州省烟草科学研究院,烟草行业分子遗传重点实验室

出　　处：《中国烟草学报》2014年第4期75-78,共4页Acta Tabacaria Sinica

基　　金：贵州省科技厅农业攻关项目(黔科合NY字[2011]3047号);中国烟草总公司重点项目(中烟办[2010]221号);中国烟草总公司重大专项(中烟办[2012]146号);贵州省优秀青年科技人才培养对象专项资金(黔科合人字[2013]02号)

摘　　要：CEL I内切酶特异识别并切断错配碱基,是建立TILLING实验技术平台的关键之一。本文从贵州省贵阳市当地种植的西芹中获得CEL I酶粗提物,利用一对具有已知单碱基差异的质粒作为底物验证所提CEL I酶粗提物的酶切活性,通过琼脂糖电泳和毛细管电泳对酶切产物进行检测,表明所获芹菜粗提物具有CEL I活性。本实验建立了适宜烟草TILLING实验的CEL I粗提物酶切体系:酶切温度42℃,酶切时间60 min,酶切反应总体积15μL,包括8μL PCR产物、4.5μL ddH2O、1.5μL酶切缓冲液(pH 7.5,500 mmol/L KCL,100 mmol/L Tris-Cl,15 mmol/L MgCl2)和1μL CEL I酶(稀释为原始粗提液0.05倍)。cellI enzyme that specifically cleaves mismatch in DNA double strands is one of the most important components of TILLING experiment platform. Crude extract of cellI enzyme was obtained from celery growing in Guiyang of Guizhou province. A pair plasmids with a known single base difference were used as substrate to verify cellI restriction enzyme activity of crude extracts, and cleavage products were detected by agarose gel electrophoresis and capillary electrophoresis, which confirmed cellI activity in crude extracts. An effective cellI digestion system was thus established： cellI enzyme effectively cut mismatch DNA in 15 μL digestion reaction solution including 8 μL PCR product, 4.5 μL ddH2O, 1.5 μL 10＆#215;cleavage buffer （pH 7.5, 500 mmol/ L KCL, 100 mmol / L Tris-Cl, 15 mmol / L MgCl2 ） and 1μL the 20 times dilution of cellI crude extraction after 60-min incubation under 42℃.

关 键 词：TILLING 芹菜CEL I核酸酶 ABI遗传分析仪 毛细管电泳 

分 类 号：Q81[生物学—生物工程]