玉米花粉特异胞质类受体蛋白激酶基因ZmSTK1和ZmSTK2累加效应调控花粉萌发与生长的机理研究

Pollen germination and pollen tube growth is not only an important physiological process during sexual reproduction in plants, but also the key research field in plant biology research. The pollen germination and pollen tube growth are usually regulated by members of the same gene family through gene function redundancy or functional differences. Our research group first found that ZmSTK1 and ZmSTK2, which coding proteins belong to pollen-specific cytoplasmic receptor-like protein kinase RLCK- IX family, regulated maize pollen germination and pollen tube growth by additive gene effects. There are no related research reports.. The present project will use GFP as a marker to determine the expression patterns of ZmSTK1 and ZmSTK2 in maize pollen germination and pollen tube growth, use the analysis of pull-down, Co-IP and bimolecular fluorescence complementation to screen substrate proteins of ZmSTK1 and ZmSTK2 for determining gene additive effect of ZmSTK1 and ZmSTK2 at the protein level; further use iTRAQ technology to clarify the signal transduction pathway regulated by ZmSTK1 and ZmSTK2 gene additive effect.. These results can not only enrich our understanding of pollen germination and pollen tube growth which are important links of sexual plant reproduction, but also be meaningful for the in-depth understanding of the controlling mechanism of plant fertilization and reproduction.

花粉萌发与生长是植物有性生殖发育的重要生理过程，是植物生物学研究重点。同一基因家族成员大多以功能冗余或功能差异模式调控花粉萌发和生长。ZmSTK1和ZmSTK2为花粉特异胞质类受体蛋白激酶RLCK-Ⅸ基因家族成员，通过基因累加效应调控玉米花粉萌发和生长，目前尚未有相关报道。. 本项目利用GFP标记确定ZmSTK1和ZmSTK2在玉米花粉萌发和生长过程中基因表达模式；利用pulldown、Co-IP及双分子荧光互补分析筛选ZmSTK1和ZmSTK2靶蛋白，明确ZmSTK1和ZmSTK2累加效应在蛋白水平上作用模式；进一步利用iTRAQ技术解析ZmSTK1和ZmSTK2基因累加效应调控玉米花粉萌发和生长的信号转导途径。探索同一基因家族成员通过基因累加效应影响花粉萌发和生长的新型调控模式，不仅丰富我们对植物有性生殖重要环节的理解，也对深入了解植物受精及繁殖的控制机理具有重要意义。

花粉萌发与生长是植物有性生殖发育的重要生理过程，同时涉及细胞识别及信号转导等生物学研究的共性问题，因而是植物生物学研究的重点。课题组从Ac/Ds插入突变体库和Mu插入突变体库中，分别筛选到花粉特异胞质类受体蛋白激酶基因ZmSTK1和ZmSTK2。前期研究发现，ZmSTK1和ZmSTK2在玉米成熟花粉中特异表达，均编码具普通胁迫蛋白受体结构域（Usp）的RLCK-Ⅸ类花粉特异胞质类受体蛋白激酶。zmstk1和zmstk2单突变体花粉萌发时间晚于野生型，且zmstk1突变体花粉萌发率约53%，导致自交结实率约30%；zmstk2突变体花粉萌发率约65%，导致自交结实率约50%；zmstk1/zmstk2双突变体自交几乎不结实。将单突变体zmstk1与zmstk2杂交获得F1代，F1自交后代穗数分离比为9:6:1（单穗结实率90%以上：约30或50%：10%以下），符合基因累加效应遗传规律。本研究首先明确了ZmSTK1和ZmSTK2基因在玉米花粉和萌发的花粉管中表达；其次，筛选了激酶ZmSTK1和ZmSTK2调控的下游靶蛋白并确定ZmSTK1和ZmSTK2与玉米烯醇化酶1和烯醇化酶2互作；第三，应用iTRAQ技术解析了ZmSTK1和ZmSTK2调控玉米花粉萌发与生长的信号转导途径，结果显示突变体玉米花粉中糖酵解途径受到影响；最后，对突变体玉米花粉中可溶性糖、丙酮酸、磷酸烯醇式丙酮酸、ATP等物质的含量进行了定量分析，结果表明突变体花粉中上述物质的含量降低。以上结果显示，ZmSTK1和ZmSTK2通过调节烯醇化酶活力调控花粉能量代谢参与花粉发育调控。本研究相关结论不仅丰富我们对植物有性生殖重要环节的理解，也对玉米花粉败育材料的创制提供了新的思路。