含有His 5基因用于酵母人工染色体重组筛选的置换型载体的构建  

Construction of a Replaced Vector with His 5 Gene for Screening of Yeast Artificial Chromosome Recombination

作　　者：任立明[1] 邹贤刚 Marianne Brüggemann 

机构地区：[1]中国农业大学农业生物技术国家实验室,北京100094 [2]Laboratory of Genetic and Developmental Immunology,The Babraham Institute,Cambridge CB2 4AT,UK

出　　处：《农业生物技术学报》2004年第2期170-174,共5页Journal of Agricultural Biotechnology

基　　金：农业部"948"项目(No.CTEC9820660A016)资助。

摘　　要：人工酵母染色体(YAC)可以克隆和分析大片段的染色体DNA,并且可以分离那些在大肠杆菌(Escherichiacoli)中不可能得到的序列。实验将His5基因插入到一个Ura3基因的中间,构建了一个新的质粒pLRH33,从而打断了Ura3 基因使之不能表达。利用His5基因两端各留下的部分Ura3基因片段,同源重组到YAC臂上原有的Ura3基因中,使重组后的YAC的标记性状从Ura+ His- 变成了His+ Ura-。用质粒pLRH33的5.5 kb线性片段以一定比例和需要重组到YAC上的目的基因pBAC300的50 kb片段相混合,同时作酵母菌转化,在His- Ura+培养基上得到大量带His5基因的阳性克隆。在检测的1 200个克隆中,有250个目的基因阳性克隆,占受检克隆的22.5%。表明质粒pLRH33可以有效地用作YAC重组的筛选标记。Yeast artificial chromosomes (YACs) can be used to clone and analyze large segments of genomic DNA and permit the isolation of sequences which are impossible to maintain in Escherichia coli. In this experiment, His 5 gene was inserted into Ura 3 gene and constructed a new plasmid pLRH33, which contained a complete His 5 gene and partial Ura 3 gene at the ends of His 5 gene. The new plasmid could be integrated into the original Ura 3 gene in the arm of YAC by homologous recombination and changed the selective marker. The plasmid pLRH33 and the insert fragment which was going to be recombined into YAC were mixed in the certain proportion and transformed into yeast, and the positive clones were selected on the medium with His- Ura+. In detected 1200 clones, 250 of which were the positive clones with the insert fragment, about 22.5% of the total detected clones. This results showed that the plasmid pLRH33 was effective for selection of YAC recombination.

关 键 词：His5基因 人工酵母染色体 重组 置换型载体 克隆 

分 类 号：Q78[生物学—分子生物学]