拟南芥雄性不育突变体atk1-7的遗传定位与功能分析

Genetic mapping and functional analysis of Arabidopsis thaliana male sterile mutant atk1-7

拟南芥ATK1是一种微管依赖性的驱动蛋白,在有丝分裂和减数分裂纺锤体组装过程中发挥着重要作用。本研究利用正向遗传学在拟南芥Ler背景下筛选到一个育性缺陷突变株。与野生型相比,该突变体营养生长不受影响,果荚短小无种子。亚历山大红染色揭示其花粉完全败育。甲苯胺蓝染色发现其四分体形成异常,出现多核现象,暗示减数分裂过程异常。遗传分析表明该突变为单基因隐性突变。利用图位克隆将目的基因定位于4号染色体上约60 kb区间。通过分析该区域内的基因,发现已报道减数分裂相关基因ATK1位于该区间内,确定为潜在目的基因。通过基因组测序发现,在该突变体中ATK1基因的第8个外显子区域有一个28 bp的缺失,产生一个丢失Motor结构域的截短蛋白。异等位杂交实验证实了该突变基因为ATK1,并把这一新突变体命名为atk1-7。利用DAPI染色和着丝粒荧光原位杂交技术分析减数分裂过程中的染色体行为,发现同源染色体和姐妹染色单体的分离过程均存在异常。利用转基因互补实验首次发现单独的ATK1蛋白Motor结构域能够恢复突变体的不育表型,表明Motor结构域具有极其重要的功能。

In Arabidopsis, ATK1 is a microtubule-dependent kinesin that plays an important role in mitotic and meiotic spindle assembly processes. In this study, we used forward genetics to screen a fertility-defective mutant under Ler background, showing that vegetative growth was not affected and the silique was short and seedless. Alexander staining revealed complete sterility of pollen. Toluidine blue staining re-vealed abnormal formation of tetrads and showed multinuclear phenotype. It suggested that the meiosis process is abnormal. Genetic analysis showed it was a single-gene recessive mutation. By map-based cloning, the target gene was located in the interval of approximately 60 kb on chromosome 4. By analyzing the genes in the interval, we found that the reported ATK1 was within this interval, and then identified it as a candidate gene. By Sanger sequencing, a 28-bp deletion in the 8 th exon of the ATK1 was found in this mutant and a stop codon was formed, resulting in a truncated protein and lacking 438 amino acids(including the entire Motor domain). Subsequently, the target gene was confirmed to be ATK1 by using transheterozygous hybridization experiment, and the new mutant was named as atk1-7. Analysis of chromosomal behavior during meiosis using DAPI staining and Fluorescence in situ hybridization, It was found that there are abnormalities in the separation of homologous chromosomes and sister chromatids. We used the transgenic complementation experiment to find that the ATK1 protein Motor domain can restore the mutant’s sterility phenotype for the first time, indicating that the Motor domain has extremely important functions.