拟南芥盐敏感突变体EMS85的快速基因定位与分析

Rapid genetic mapping and analysis of a salt-sensitive mutant EMS85 in Arabidopsis thaliana

土壤盐碱化是造成农作物减产的主要原因.作者通过对拟南芥(Arabidopsis thaliana)野生型种子Col-0进行甲基磺酸乙酯(EMS)诱变,筛选到1株盐敏感突变体EMS85.通过杂交和后代性状分离比确定该突变体的不耐盐性是受隐性单基因控制.采用图位克隆的方法,通过对拟南芥5条染色体上的SSLP标记的筛选,挑选了24对均匀分布于染色体上的SSLP标记作为基因初定位,同时设计了一系列精细定位分子标记,通过粗定位和精细定位发现,该基因位于拟南芥第5条染色体下臂的分子标记LUGSSLP847和5-13.58之间,进而测序结果表明该突变体是SOS2基因的等位突变体,突变位点为该基因1 521~1 522 bp处两碱基的缺失,造成移码突变,使SOS2基因功能丧失.本实验室建立的图位克隆体系加快了克隆基因的进程,对EMS85突变体的定位只在短短半年时间就已完成,为其他突变体的快速确定候选基因,加速基因分离进程奠定了基础.

Sah stress is one of the major factors reducing crop yield.In this study,an Arabidopsis salt-sensi- tive mutant EMS85 was obtained by ethyl methane sulphonate （EMS） mutagenesis strategy.Genetic analysis indicated that salt-sensitive phenotype was controlled by a single recessive locus. Using map-based cloning technique ,24 pairs of SSLP markers were picked up which evenly distributed on the 5 chromosomes as the coarse mapping, and a series of molecular markers were designed through the fine mapping.Through coarse mapping and fine mapping,EMS85 has been mapped to a region between molecular marker LUGSSLP847 and 5-13.58 on the chromosome V.The mutant was proved to he an allelic mutant of SOS2 gene by sequencing and blasting, mutational site carried a two deletion in the 1 521--1 522 bp of SOS2 gene, which caused frameshift mutation and functional incapacitation of SOS2 gene.The map-based cloning technique has greatly accelerated the process of gene cloning, it takes half a year in localizing the EMS85mutant, which could determinate a candidate gene quickly for other mutants and accelerate the gene mapping process.