拟南芥AtFRO1基因调控减数分裂同源染色体重组的分子机制

Meiotic recombination is not only required for the accurate segregation of homologous chromosomes during meiosis I, ensuring the functional gamete formation, but increases genetic variations among daughter cells due to chromosome exchanges. Therefore, comprehensively understanding the molecular mechanism of meiotic recombination is very important for both reproductive biology and genetic breeding. Through high-throughput genetic screening, we found one highly interesting mutant, named Atfro1, with distinct phenotypes in fertility defects and meiotic chromosome behaviors. At early reproductive stage, the mutant is completely sterile. However, at late stage, its fertility will be gradually recovered, even fully restored. Chromosome behaviors analyses indicated that during the transition from metaphase I to anaphase I, plenty of fragment chromosomes were observed. Those novel phenotypes suggest that AtFRO1 is a potentially new gene involved in meiotic recombination. Further genetic and sequencing mapping data confirmed this conclusion. In this project, we are planning to integrately use approaches in genetics, molecular and cellular biology, immunology, genomics and transcriptomics to clone and analyze AtFRO1 function, revealing the molecular mechanism regulated by AtFRO1. More importantly, benefiting from the special phenotype of fertility restored at late stage, we obtained the unique opportunity to investigate the possible remodelling mechanism of meiotic recombination in multiple cellular eukaryotes.

减数分裂同源染色体重组既保证了同源染色体的正确分离和功能配子的正常形成，又增加了配子之间的遗传多样性。因此，深入理解减数分裂同源染色体重组的分子机制，对于生殖发育学和遗传育种学都有重要意义。本项目通过筛选拟南芥突变体发现了一个在育性和同源染色体重组方面表型都十分新颖的突变体，命名为Atfro1。该突变体在生殖发育前中期表现为完全不育，到后期育性则恢复。染色体行为分析表明突变体在中期I向后期I转变过程中出现片段化染色体。以上两种表型还未见在已有减数分裂突变体中报道，暗示AtFRO1为一新重组基因，且初步遗传定位分析也支持这一结论。本项目拟采用遗传学、分子和细胞生物学、免疫学、基因组学和转录组学等方法，首先对AtFRO1基因的定位和功能进行全面研究，以揭示其参与减数分裂同源重组的分子机制；进而以突变体后期育性恢复这一特殊表型为契机，综合研究多细胞真核生物减数分裂同源重组可塑性的分子调控机。

减数分裂同源染色体重组是真核生物配子体形成所必须的，为遗传多样性的产生提供物质基础。在本项目研究中，我们以模式植物拟南芥为研究材料，通过筛选Ac-Ds插入突变体库获取一个表型独特的突变体，被初步命名为AtFRO1。进而通过全基因组测序和图位克隆以及异等位杂交等实验证明AtFRO1为蛋白激酶ATM的一个等位突变，因此命名该等位突变为atm-4。序列分析发现该突变体中有一个约700bp的DNA片段插入，最终可能导致形成一个缺失ATM激酶结构域的残缺蛋白。通过比较多个ATM等位突变体，我们发现atm-4表型最为严重。免疫荧光实验证明联会复合体蛋白ASY1和ZYP1在atm中的定位没有发生变化，而且其突变也不影响粘连蛋白SYN1的定位。然而我们发现在atm中γH2AX的信号减少，但减数分裂DSB的数目却增加。同时利用分析HEI10在染色体上的定位，我们发现atm突变体中I型CO的形成收到影响。通过构建双突变体分析减数分裂染色体动态变化，我们发现ATM可能促进RAD51介导的依赖姐妹染色单体修复的途径。此外，我们利用转录组比较分析了野生型和突变体减数分裂细胞中基因表达水平的变化，鉴定出多个受ATM影响的基因，为后期研究奠定了良好的基础。总之，该课题深入研究了ATM在减数分裂同源染色体重组中的作用，为揭开ATM介导的减数分裂DSBs修复机制提供了重要依据。