染色体上大片段DNA的直接克隆及移植  

Direct Cloning and Transplanting of Large DNA Fragments from Escherichia coli Chromosome

作　　者：朱莹[1,2] 杨艳[1,2] 邓平平[1,3] 黄勇[4] 倪孟祥[2] 方宏清[1,3] 

机构地区：[1]中国人民解放军军事医学科学院生物工程研究所,北京100071 [2]中国药科大学生命科学与技术学院,南京210009 [3]安徽大学健康科学研究院,合肥230039 [4]中国人民解放军军事医学科学院微生物与流行病研究所,北京100071

出　　处：《中国科学：生命科学》2016年第2期183-191,共9页Scientia Sinica(Vitae)

基　　金：国家重点基础研究发展计划(批准号:2011CBA00800);国家自然科学基金(批准号:81373286)资助

摘　　要：应用Red重组体内直接克隆染色体上大于10 kb的DNA序列非常困难.利用一种抗性拆分-融合策略成功地从供体菌染色体上直接克隆了约50 kb的DNA序列,并将其移植到受体菌的染色体上.首先将抗性基因(如cat)的互补片段cmb定向整合到染色体上拟克隆区域的一侧,然后将含有抗性基因另一互补片段cma的线性质粒载体电转到细胞内进行定向捕获,通过抗性筛选获得克隆.使用该方法,成功地将供体菌DH1-Z基因组上约48kb的大片段DNA捕获至质粒载体上,捕获阳性率达80%.然后,将该质粒转化到受体菌DH36,利用双链断裂促进同源重组策略,替换受体菌染色体上对应区域,一次敲除基因组上4个非必需区,获得重组菌DH40.该方法也可用于微生物天然产物生物合成途径的异源表达、基因组组装和大片段DNA的功能研究.We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts： cma and cmb（has a homologous sequence with cma）. Each of them has no resistance separately unless the two parts are fused together. The cmb fragment was first integrated into a side of the target cloning region, then a linear cma-containing plasmid vector was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned a DNA fragment about 48 kb from E. coli DH1-Z chromosome with positive frequency of about 80%. Then, combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to replace the corresponding region of E. coli DH36 chromosome and knock out four nonessential genomic regions at one time. This strategy provides a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways, for genome assembly and for the function study of large DNA sequence.

关 键 词：RED同源重组 抗性拆分-融合 直接克隆 移植 

分 类 号：Q78[生物学—分子生物学]