激活标签法构建拟南芥突变体库及其表型分析

Constructing and Phenotypic Analyzing an Activation-Tagging Arabidopsis Mutant Pool

以拟南芥(Arabidopsis thaliana)野生生态型(Columbia)植株为实验材料,以含有激活标记双元质粒pCB260的农杆菌进行转化,并以抗除草剂Basta为筛选标记,构建了拟南芥激活标签突变体库,所用pCB260双元质粒含有两个Ds位点、一个GFP标记基因与一个抗basta标记基因,可以方便高效地筛选转基因植物。目前经初步筛选获得了约10000个独立转化株系(T1代),其中约50个株系具有明显的表型变化,包括花期改变、株型变异、叶形特异、育性降低、花发育异常、种子颜色变浅等。运用TAIL-PCR技术,成功获得了其中10个表型特异株系的T-DNA侧翼序列,分别分布于拟南芥基因组的5条染色体上。

Activation tagging method is an effective tool of obtaining gain-of-function mutant and investigating the gene function,which plays an important role in plant functional genomics study. In this paper,we used Arabidopsis Columbia wild type as material to construct an activation tagging mutant pool by Agrobacterium tumefaciens-mediated transformation,the binary vector pCB260 contained two Ds elements,one GFP report gene and one basta resistance selection genes,which show more convenient and efficient to screen the transgenic plant. Until now,over ten thousand transformed plants were generated. Among them,about 50 dominant mutants with obvious phenotypes were isolated,including early or late flowering time,unmoral leaf shape and flower,sterility and thin seed capsule color. T-DNA flanking sequences of ten special mutants were validated by TAIL-PCR and sequencing,whose T-DNA insertion fragments distributed in all five chromosomes of Arabidopsis genome,respectively.