【作者】 池正昌；

【导师】 陈建民； 程祝宽；

【作者基本信息】 扬州大学 ， 细胞生物学， 2010， 硕士

【摘要】 减数分裂是有性生殖物种世代交替的转折点;减数分裂不仅维持了基因组的稳定性,而且通过重组创造了遗传多样性。在减数分裂过程中,DNA复制一次后进行两次连续的细胞分裂。第二次减数分裂与有丝分裂类似,涉及到姊妹染色单体的分离;而减数第一次分裂是独一无二的,在这次分裂中同源染色体分离。为保证同源染色体的准确分离,同源染色体在减数分裂前期I通过一系列复杂的过程结合在一起形成稳定的二价体。这些复杂的前期I事件包括同源配对、联会和重组。分子遗传学、细胞学和生物化学研究已经表明玉米Sgo1基因在减数分裂过程中起到了保护着丝粒处黏着蛋白的重要作用。OsSGO1基因是玉米Sgo1基因在水稻中的同源基因。本课题应用RNA干扰技术分析了该基因在水稻生长发育过程中的功能。利用本实验室设计的RNAi工具载体pCam23A构建OsSGO1-RNAi载体;并通过农杆菌介导的水稻愈伤组织转化法获得了转基因株系;进而通过PCR鉴定筛选出阳性的OsSGO1-RNAi株系。OsSGO1-RNAi株系的营养生长正常,但结实率显著降低;通过内源OsSGO1基因表达量的半定量RT-PCR证实OsSGO1-RNAi株系的不育表型与RNA干扰介导的内源OsSGO1 mRNA水平的降低是相关的。进一步深入的细胞学观察显示,OsSGO1基因的knockdown导致OsSGO1-RNAi株系的减数分裂异常,具体表现为姐妹染色单体在末期Ⅰ时提前解离,后期Ⅱ时不均等分离,最终导致花粉败育;Immunostaining实验揭示OsSGO1蛋白定位于着丝粒处。这些研究结果表明OsSGO1基因是水稻减数分裂的必需基因,该基因在减数分裂过程中对姐妹染色单体着丝粒处的黏着起了重要的保护作用。更多还原

【Abstract】 Meiosis, an essential process in sexual life cycle, is necessary not only for the maintenance of genomic stability but also for the creation of genetic diversity. Meiosis involves two sequential rounds of chromosome segregation following a single round of DNA replication. The first meiotic division is unique and involves the segregation of homologous chromosomes. To ensure accurate segregation, homologous chromosomes form stable bivalents through a complex processes which include homologous pairing, synapsis and recombination. The corn Sgo1 gene has been shown to play an important role in protecting of centromeric cohesion. OsSGO1 is the rice homologue of the maize Sgo1 gene. Here, we analyzed the function of OsSGO1 in meiosis using the RNA interference (RNAi) approach. We constructed an OsSGO1-RNAi vector and transformed it into rice calli by Agrobacterium-mediated DNA transfer. OsSGO1-RNAi lines were obtained by screening the plants regenerated from the transformed calli by PCR. The OsSGO1-RNAi lines showed normal vegetative growth but sterile during flowering phase. The sterility phenotypes were associated with down-regulated OsSGO1 transcript level mediated by RNAi. Further cytological observations of male meiocytes revealed that knockdown of OsSGO1 caused precocious disassociation and random segregation of sister chromatids at telophaseⅠand anaphase II, respectively, which finally led to sterile pollen formation. Immunostaining experiments revealed that the OsSGO1 localizes to centromere. These results indicate that OsSGO1 is essential for rice meiosis and plays an important role in protecting centromeric cohesion during meiosis.更多还原