万寿菊雄性不育关键基因TePI的转录调控研究

Spontaneous homeotic conversion of floral organs was the underlying cause of the male sterility in marigold (Tagetes erecta).Thus, petals of male sterile marigold resembled sepal-like structures and the stamens were partially converted to styles. We discovered that decreased expression level and mutation of gene structure of TePI gene were the major reasons of floral organs’ homeotic mutation in male sterile marigold. On one hand, to clarify the cause of decreased expression levels of TePI gene, the upstream transcription factors are identified and analyzed. Firstly, promoter cloning and promoter deletion are used to make clear the efficient transcription site and transcriptional efficiency of TePI and Tepi genes’ promoter. Secondly, yeast one-hybrid library screening technology is conducted to find out the upstream transcription factors of TePI and Tepi. Thirdly, Yeast one-hybrid technology (point to point), sequence analysis, quantitative RT-PCR and protoplast transformation are adopted to test the direct transcription factor. Finally, subcellular localization and genetic transformation are used to clarify the action site and function of target transcription factors. On the other hand, a series of modern experimental techniques, such as, Semi-quantitative RT-PCR, quantitative RT-PCR, yeast hybrid assay, bimolecular/trimolecular fluorescence complementation and genetic transformation, are adopted to study the effect of the gene structure mutation of TePI on the expression level and protein interaction of downstream targeting genes. The implementation of the project can enrich the understanding of the molecular mechanism of male sterile plants. The creation of the artificial male sterile material, to improve the breeding efficiency, has potential application value.

雄性不育万寿菊的花器官发生同源异型突变导致花瓣萼片化，雄蕊柱头化，是非常理想的育种亲本。前期研究表明万寿菊TePI基因表达量下降和结构突变是导致花器官同源异型突变的关键原因。为阐述导致TePI基因表达量下降的原因，项目首先通过启动子克隆和分段技术明确TePI/Tepi基因的启动子片段在植物体内的有效转录部位和转录效率；采用酵母单杂交筛库技术、序列分析、qRT-PCR和原生质体转化等确定直接调控TePI/Tepi的转录因子；最后通过亚细胞定位和遗传转化明确目标转录因子作用部位和功能。另一方面项目采用半定量RT-PCR、qRT-PCR、酵母杂交和双/三分子荧光互补、遗传转化等实验技术，从表达和蛋白互作等方面研究TePI基因结构突变对下游受控MADS-box基因的影响。项目的实施可以丰富对植物雄性不育分子基础的认识， 对人工创建雄性不育材料进而提高育种效率具有潜在的应用价值。

项目背景：万寿菊为菊科植物，复杂的头状花序增加了人工去雄的困难，阻碍杂了交育种的工作进程。本课题组前期研究表明万寿菊雄性不育株的花器官发生了同源突变，即花瓣突变为萼片结构，雄蕊突变成柱头状。遗传学分析发现雄性不育植株中的class B类基因tepi发生了突变，其在第四个外显子处插入一个1088bp长度的序列，导致其蛋白质序列多出3个氨基酸。根据TePI和tepi之间序列差异开发的标记能够成功的应用于万寿菊雄性不育系F2杂交群体中。.主要研究内容：我们利用BSR技术挖掘雄性不育的候选基因，并利用生物学技术从基因的表达模式、蛋白互作模式以及TePI和tepi基因的功能等角度上解析万寿菊雄性不育的机理。.重要结果和关键数据：（1）通过三代转录组和BSR-seq技术定位到TePI基因是万寿菊雄性不育的关键基因。（2）表达分析表明，TePI突变为tepi后，其他的MADS-box A，B，C，D，E类基因在不育株系和可育株系的花器官和组织中的表达模式发生变化。（3）转pi突变的拟南芥，表现观察显示TePI可以使突变体植株恢复可育，而tepi却未能使突变体植株恢复可育。 此外，异源转化野生型拟南芥结果表明单独转化tepi 和TePI 基因均未能影响转基因植株的生长发育，但同时将TePI和TeAP3-2转入拟南芥中导致转化苗的莲座叶数和抽薹数显著增加，而同时将tepi和TeAP3-2转入拟南芥中却未能影响转化苗的莲座叶数和抽薹数。表明，Tepi基因的功能缺失。（4）酵母双杂和BIFC实验结果表明TePI/tepi蛋白均可以与class B类蛋白形成同源或者异源二聚体，而与class E类蛋白互作中验证中发现tepi蛋白可以与TeSEP4蛋白互作，而TePI蛋白不与TeSEP4蛋白互作。酵母三杂实验中进一步发现TePI和tepi通过与TeAP3-2形成异源二聚体的形式与C， E类蛋白互作，其中TePI+TeAP3-2可以与TeSEP3-3形成三聚体复合物，而tepi+TeAP3-2不能与TeSEP3-3互作。表明tepide 蛋白互作模式发生变化，从而影响了蛋白的功能。.科学意义：增进了对花器官同源异型突变分子机理的认识，为人工创造花瓣退化、雄蕊柱头化的不育材料提供理论支持。