番茄DNA复制因子RFC1在减数分裂重组中的功能研究  

Functional Analysis of the Tomato DNA Replication Factor RFC1 in Meiotic Recombination

作　　者：刘静[1] 王英[1] 王宏宽[1] 倪迪安[2] 马红[1] 王应祥[1] 

机构地区：[1]复旦大学生命科学学院植物科学研究所,上海200433 [2]上海应用技术学院生态技术与工程学院,上海201418

出　　处：《复旦学报（自然科学版）》2016年第5期623-631,641,共10页Journal of Fudan University：Natural Science

基　　金：国家自然科学基金面上项目(31570314);荷兰Rijk Zwaan种业公司项目

摘　　要：减数分裂是真核生物有性生殖所必需,前期研究表明拟南芥DNA复制因子RFC1在减数分裂重组中具有重要作用,但其同源基因在其他物种中的减数分裂或其他功能还未有报道.为了检验番茄RFC1在减数分裂中的功能,我们建立了减数分裂特异RFC1^(RNAi)转基因番茄,发现其花粉活性显著降低,而且减数分裂异常.利用DAPI染色观察染色体形态,发现RFC1^(RNAi)减数分裂细胞在终变期和中期Ⅰ形成多价体,表明重组异常.DNA双链断裂指示蛋白γH2AX在RFC1^(RNAi)粗线期染色体上分布的保留滞后,进一步支持RFC1^(RNAi)中的重组异常.拟南芥和番茄分别属于真双子叶植物中的两个不同大支,蔷薇类和菊类,因此本文的结果也表明DNA合成基因RFC1在进化距离很远的不同植物物种中的功能相对保守.Meiosis is essential for eukaryotic sexual reproduction.Previous study showed that Arabidopsis RFC1 is required for normal meiotic recombination,but the role of its homologs in other species has not been reported.To study the tomato RFC1 homologue in meiosis,the in vivo function of SlRFC1 was investigated by silencing the SlRFC1 gene using a meiosis-specific DMC1 promoter in transgenic RFC1 ^（RNAi） plants.Phenotypic analysis found that RFC1 ^（RNAi） plants have obviously reduce pollen viability and fertility,and are abnormal in meiosis.Comparison of chromosome morphology of WT and RFC1 ^（RNAi） meiocytes using chromosome spread with DAPI staining showed that,unlike wild type diakinesis and metaphaseⅠ meiocytes with 12bivalents（pairs of associated homologous chromosomes）,the RFC1 RNAi meiocytes formed multivalents at similar stages,indicating non-homologous association.Immunolocalization of DNA double stranded break（DSB）markerγH2AX showed persistent signals in RFC1 RNAi pachytene chromosomes compared with WT,providing further evidence for abnormal recombination in RFC1 RNAi.In conclusion,this is the first time that the tomato DNA replication factor RFC1 is shown to be required for meiotic recombination,using meiosis-specific gene silencing.Since Arabidopsis and tomato belong to two difference clades of Rosids and Asterids among eudicots,respectively,our results also reveal the RFC1 function in meiotic recombination is conserved in meiosis among evolutionarily different angiosperm species.

关 键 词：减数分裂 DNA复制因子 番茄 减数分裂特异性RNA干扰 

分 类 号：Q5[生物学—生物化学]