拟南芥激活标记突变体库的构建及突变体基因的克隆

激活标记(activation tagging)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(Arabudidopsis thaliana)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20 000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DNA Southern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(plasmid rescue)和TAIL-PCR(Thermal asymmetric interlaced-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.

Construction of an Activation Tagging Library of Arabidopsis and Cloning for Mutant Genes

Activation tagging is an important strat- egy in plant genomics by generating gain-of-func- tion mutants. In this work, a library of Arabidopsis mutants was constructed by in planta transforma- tion mediated by Agrobacterium tumefaciens con- taining 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 mor- phological phenotypic variations (Fig.2). Results of Southern blot analysis revealed that most of the transformants have more than three copies ofT-DNA insertion (Fig.3). Plasmid rescue and TAIL- PCR were used to recover the flanking genomic se- quences of mutated target genes as the first step towards mutant gene cloning (Figs.4, 5).