雄蕊发育抑制基因MINP1的分离和作用机制研究

Proper stamen development is essential for plant fertility. It shows important scientific value and application prospects to study the molecular mechanism of stamen development. The development of stamen is controlled by a complicated genetic and signaling network. Deficiency in the phytohormones jasmonate (JA) will lead to arrested stamen development and male sterility in Arabidopsis, maize and rice. JA activates the transcription factors MYB21 and MYB24 to enhance stamen development in Arabidopsis. The knowledge on positive regulators of stamen development is rapidly increasing, while negative regulators in stamen development remain largely unknown. To isolate negative regulators, we overexpressed JA-responsive genes in flower, and found that overexpression of MINP1 represses stamen development and results in male sterility. However the molecular mechanism of MINP1 in repressing stamen development remains unknown. We aim to study the developmental role of MINP1 in stamen, the expression pattern and subcellular localization of MINP1, isolate the downstream genes of MINP1 by microarray. Our primary data also showed that MINP1 interacts with MYB21 in yeast and plant, and we will investigate the interaction of MINP1 with MYB21, interference of MINP1 on the stability and transcriptional activity of MYB21, regulation of MINP1 by jasmonate pathway, and explore whether MINP1 mediates the crosstalk of jasmonate with other pathways, finally, reveal the molecular roles and mechanisms of MINP1 in repressing stamen development, which will help to broaden our knowledge on plant reproductive development.

雄蕊发育受到精致的调控。拟南芥、玉米和水稻等植物缺失茉莉素会导致雄性不育。茉莉素通过释放转录因子MYB21/MYB24促进雄蕊发育。人们对促进雄蕊发育的基因认识不断增加，但对雄蕊发育抑制基因的认识相对很少。我们前期通过超表达花中茉莉素响应基因的策略分离抑制雄蕊发育的基因，发现超表达MINP1能够抑制拟南芥雄蕊发育导致不育，但不清楚抑制机理。申请人拟明确MINP1对雄蕊的发育学影响，研究MINP1的表达模式和亚细胞定位，通过基因表达芯片获得受MINP1调控的下游基因。初步研究还发现MINP1与MYB21互作，该项目拟进一步明确MINP1与MYB21互作的机制，对MYB21蛋白稳定性和转录功能的调控，MINP1受茉莉素调控的机制，MINP1如何介导茉莉素与其它雄蕊调控通路的互作，最终揭示MINP1抑制雄蕊发育的角色和分子机制，丰富人们对雄蕊发育调控机理的认识。

植物的生长发育受到精密严格的调控。MYB21和MYB24是调控雄蕊发育的核心转录因子。当植物缺乏茉莉素时MYB21和MYB24受到抑制引起雄性不育。当茉莉素存在时，诱导JAZ蛋白降解，释放MYB21、MYB24来恢复植物育性，MYB21调控雄蕊发育植物生长发育过程的认识相对很少。.我们通过酵母双杂交筛选到与MYB21/24互作的DELLA蛋白RGA（命名为MYB21-Interaction Protein, MINP1），发现MYB21、MYB24均通过N端结构域与RGA（MINP1）等DELLA蛋白发生互作。Pull-down实验发现，MYB21、MYB24与DELLA蛋白（RGA/RGL1/RGL2）存在互作。原生质体瞬时转化实验证明，DELLA蛋白RGA/RGL2均能抑制MYB21/MYB24转录活性，进而负调控雄蕊花丝伸长。.JAZ蛋白作为茉莉素通路重要抑制因子发挥重要功能。我们通过酵母双杂交实验发现，MYB21和MYB24通过N端与JAZ蛋白家族中多个成员（JAZ1/2/3/4/5/6/8/10/11/12蛋白）发生互作。原生质体瞬时转化实验证明，JAZ蛋白不仅能抑制MYB21的转录活性，还能与DELLA蛋白共同抑制该转录因子的活性。Western Blot实验还发现JA处理会加速MYB21蛋白的降解，GA能延缓MYB21蛋白的降解，即赤霉素与茉莉酸对MYB21蛋白稳定性存在拮抗作用。.我们还筛选到与MYB21互作的转录因子IIId bHLH转录因子bHLH13、bHLH17、bHLH3和bHLH14。IIId bHLH转录因子很可能抑制茉莉素调控的雄蕊发育。酵母双杂交实验显示，bHLH13的C端介导了bHLH转录因子之间的二聚化。原生质体瞬时转化实验证明，bHLH13为转录抑制因子。超表达bHLH13会抑制拟南芥对灰霉菌和甜菜夜蛾的抗性，且茉莉素受体COI1的突变阻断了四突变体bhlh13 bhlh17 bhlh3 bhlh14（Q4）的多个茉莉素超敏反应。