植物无标记转化的诱导表达Cre/loxP重组系统的构建  

Construction of Selectable Marker-free Plant Transformation Vectors Using Inducible Cre/loxP Site-specific Recombination System

作　　者：段小瑜[1] 张录霞[1] 马超[1] 郝青楠[1] 马兵钢[1] 

机构地区：[1]石河子大学农学院,新疆石河子832003

出　　处：《石河子大学学报（自然科学版）》2008年第1期30-34,共5页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(30460081);新疆维吾尔自治区高等学校科研计划资助项目(XJEDU2005S15)

摘　　要：应用Cre/loxP系统位点专一性重组的特点构建诱导表达的定位重组系统,用以特异性的敲除转基因植物的标记基因。为了获得诱导表达启动子,从大豆基因组DNA中用pfu酶克隆热激蛋白启动子gmhsp17.5c,将其克隆到pUC118-HincⅡ载体并测序。结果表明,508nt的gmhsp17.5c与已报道序列(GenBank,AF544399)比较,核苷酸的同源性为99.8%。利用该诱导启动子分别构建了含gmhsp17.5c-cre基因组件和gmhsp17.5c-gus基因组的诱导型植物表达载体pC23HC和pC23HG。此外1个含有loxP-gus-loxP组件的组成型植物表达载体pC23LG被构建。通过对3个植物表达载体做多重酶切及亚克隆后测序分析表明载体pC23HC全长10947bp,载体pC23HG全长11396bp,载体pC23LG全长11900bp,符合预期设计。一套由pC23HC和pC23LG组成的植物无标记转化的诱导表达Cre/loxP重组系统被构建,为进一步将诱导表达的标记基因删除系统用于植物的无标记转化奠定基础。Cre/loxP site-specific recombination system was used to constmct plant expression vectors for mark-free transgenic plant.The inducible promoter of grnhsplT. 5c gene was amplified by polymerase chain reaction from genomic DNA of Glycine max L. and it was sequenced after being cloned into pUC118-Hinc Ⅱ vectors. The sequencing results indicated that the cloned fragment of gmhsp17. 5c contained 508 nucleotides, shared a sequence homology of 99.8% with that from GenBank accession number AF544399. Using this promoter, the inducible plant expression vector pC23HC of 10947 bp and pC23HG of l l396bp were constructed including grnhsp 17.5c-cre and grnhsplT. 5c-gus sequences separately, while another plant expression composition vector pC23LG of 11900 bp including loxP-gus-loxP boxes was constructed. The selectable marker-free plant transformation vectors were constructed using inducible Cre/loxP site-specific recombination system to be used into marker-free transgenic plants. The Agrobacterium-mediated transformation was conducted for the base use of further research of the knockout system.

关 键 词：Cre/loxP定位重组系统 标记基因 载体构建 热激启动子 

分 类 号：S565.4[农业科学—作物学] Q782[生物学—分子生物学]