【作者】 王晨；

【导师】 杨仲南；

【作者基本信息】 上海师范大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 AtMYB103是R2R3-MYB家族的转录因子,调控着拟南芥花药的正常发育和花粉的形成。该基因敲除突变体ms188的花药绒毡层细胞发育异常,四分体无法正常释放,花粉外壁结构缺失,最终导致花粉的败育。在前期工作的基础上,本文对AtMYB103的调控花药发育的分子机理展开更深入的研究。亚细胞定位结果显示AtMYB103最终在细胞核中行使其调控下游基因转录的功能;同时,我们通过酵母双杂交技术阐明在转录因子AtMYB103的C端62个aa区域中存在着具有转录激活功能的结构域,与N端的R2R3区共同构成了其转录调控的基本功能结构。基于前期工作中初步确定绒毡层发育主要基因的调控关系,通过荧光实时定量PCR及上游基因启动子驱动的下游基因转入上游基因突变体的手段来进一步验证。为了研究AtMYB103的相互作用基因及上下游的调控基因,我们构建了花药特异表达基因的酵母杂交cDNA文库,此外,我们还获得了AtMYB103与TAP标签融合的转基因植株,这些工作为利用CHIP技术寻找在体内被AtMYB103直接调控转录的基因,进一步研究AtMYB103相关的花药发育分子调控机理奠定了基础。更多还原

【Abstract】 AtMYB103 encodes a R2R3-type of MYB transcription factor which regulates normal anther and pollen development.The knock-out muant of AtMYB103,ms188,showed defective tapetum development,altered callose dissolution and surviving microspores lacked exine,resulted in male sterile phenotype.Base on the previous works, investigation of AtMYB103 in this study improve comprehension of the mechanism of anther development.The subcellular localization showed that AtMYB103 protein is localized to the nucleus;we identified that the activation domain was localized in 62 residues at C-terminus of AtMYB103 by yeast two-hybrid,which compose the functional structure of transcription factor with R2R3 DNA binding domain at N-terminus.Base on the primary regulatory network of tapetum development, the quantitive RT-PCR was performed to confirm the results.We further constructed the downstream gene driven by upstream promoter to determine the relationship of these key regulator of tapetum。For screening the interacting proteins of AtMYB103 and up-/downstream genes,we constructed the yeast hybrid library of Arabidopsis inflorescences.Furthermore,the transgenic line of AtMYB103 and TAP-tag fusion protein was obtained,which provide easy access to find the direct target of AtMYB103 by CHIP.These works lay the foundation for further investigation of the molecular basis of AtMYB103 in anther development.更多还原