拟南芥激活标记突变体库的构建及突变体基因的克隆  

Construction of an Activation Tagging Library of Arabidopsis and Clon- ing for Mutant Genes

作　　者：李志邈[1] 张海扩[2] 曹家树[1] 何祖华[2] 

机构地区：[1]浙江大学蔬菜研究所 [2]中国科学院上海生命科学研究院植物生理生态研究所植物分子遗传国家重点实验室,上海200032

出　　处：《植物生理与分子生物学学报》2005年第5期499-506,共8页Journal Of Plant Physiology and Molecular Biology

基　　金：国家自然科学基金项目(No.30125030)资助~~

摘　　要：激活标记(activationtagging)技术是以功能获得突变体为研究对象,在植物功能基因组学的研究中具有重要的作用。文章以双子叶模式植物拟南芥(Arabidopsisthaliana)野生生态型植株为实验材料,以含有激活标记质粒pSKI015的农杆菌直接喷雾进行转化,并以抗除草剂Basta为筛选标记,构建了拟南芥的激活标记突变体库。结果共得到约20000个独立转化株系(T1代),其中38个株系有明显的表型变化,约占转化植株总数的千分之二。基因组DNASouthern杂交结果表明,大多数转化植株为多拷贝T-DNA插入。通过质粒拯救(plasmidrescue)和TAIL-PCR(Thermalasymmetricinterlaced-PCR)可获得T-DNA插入的基因组旁邻序列,为克隆突变体的基因奠定基础。Activation tagging is an important strategy in plant genomics by generating gain-of-function mutants. In this work, a library of Arabidopsis mutants was constructed by in planta transformation mediated by Agrobacterium tumefaciens conraining an activation tagging vector pSKI015 with herbicide Basta as a selection marker （Fig. 1 ）. Among 20 000 independent transformants, 38 lines, i.e. about 0.2% of T1 progeny, show visible morphological phenotypic variations （Fig.2）. Results of Southern blot analysis revealed that most of the transformants have more than three copies of T-DNA insertion （Fig.3）. Plasmid rescue and TAIL- PCR were used to recover the flanking genomic sequences of mutated target genes as the first step towards mutant gene cloning （Figs.4, 5）.

关 键 词：激活标记 功能获得 拟南芥 质粒拯救 TAIL—PCR 

分 类 号：Q943.2[生物学—植物学]