pYTAC747HsacB载体的培养与功能分析.docVIP

pYTAC747H/sacB载体的培养与功能分析 *，肖炎凤，吴东旺，叶玉平，谭啸，彭迪 （湖南工程学院化学化工学院, 湖南 湘潭 411104） 摘要：通过对pYLTAC747H/sacB载体进行优化培养，绘制其生长曲线，用碱裂法小量提取TAC载体DNA，琼脂糖凝胶电泳分离载体DNA，研究了菌体活性及pYLTAC747H/sacB载体的一系列性能，获得了pYLTAC747H/sacB载体的一系列数据，可以作为载体pYLTAC747H/sacB培养条件的优化，标记筛选等的依据。pYTAC747H/sacB，大肠杆菌DH5α，卡那霉素ulture and Functional Analysis of pYTAC747H/sacB Vector Cao Hui Xu Yueyu* Xiao Yanfeng Wu Dongwang Ye Yuping Tan Xiao Peng Di (Chemistry and Engineering Department of Hunan Engineering Institute, Xiangtan 411104, China) Abstract: By culture and functional analysis of pYTAC747H/sacB vector，we can received a series of data of vector pYTAC747H/sacB,the series of data can be a Optimization of Culture Conditions or marking the basis for selection to pYTAC747H/sacB. By using LB culture medium as a basis for its revitalization, culture bacteria，By measuring its absorbence to draw the growth curves of E. coli DH5α which carry vector pYTAC747H/sacB. It’s proved that pYTAC747H/sacB have the resistance of kanamycin by the method of Contrast culture. A small amount of extracting plasmid DNA in a alkalic condition，Agarose gel electrophoresis separation of plasmid DNA, have researched the activity of bacterial and a range of performance of pYLTAC-747H/sacB vector.Key words: pYTAC747H/sacB E. coli DH5α Kanamycin Liu YG 等结合PAC(PI-derived artificial chromosome)载体和BIBAC(a- binary BAC)载体的特点，构建了植物可转化人工染色(Transformation- compe tent artificial chromosome,TAC)载体。TAC载体含Pl噬菌体和发根农杆菌Ri质粒的复制子，从而它能够在大肠杆菌和根癌农杆菌中以单拷贝形式穿梭复制(樊颖伦等，2004; Kotani等，1998) 【3】。因此，一旦筛选到目标阳性克隆，可直接利用农杆菌转化系统将其转到植物体中进行基因功能互补实验，不必进行亚克隆，减少了工作量，加速了工作进程。此外TAC载体还含有一个卡那霉素的抗性基因，便于进行目的基因的筛选。 pYTAC747H/sacB的物理图谱如下图所示【3】： pYTAC747H/sacB的物理图谱 1、材料与方法 1.1、菌种 E. coli DH5α，pYTAC747H/sacB：由华南农业大学的刘耀光科学家提供。 1.2、培养基及试剂 1.2.1、LB液体培养基（组分为百分比/%） 蛋白胨（Bacto tryptone）1%，酵母提取物（Bacto yeast extract）0.5% ，NaCl(sodium chloride) 1%。 1.2.3、LB固体培养基（组分为百分比/%） 蛋白胨1%，酵母提取物0.5% ，NaCl1%。加入1.5%的琼脂（Agar)配成固体培养基。 以上的培养基均调节PH至7.0并添加过滤除菌的卡那霉素，至终浓度25μg/ml 1.2.4、碱法提取质粒用试剂的配置 溶液I：50 mmol／L葡萄