水稻甲基转移酶基因OsTSD2的克隆及其对种子萌发的调控机制研究

Seed germination is a critical process in the life cycle of seed plant and is the starting point for important crop production, so detection of its mechanism does have both theoretical and practical significance. To explore the mechanisms of rice seed germination, a mutant which shows delayed seed germination was acquired by screening in a T-DNA insertion mutant library. The flanking sequence cloning and identification showed that the mutation locus is OsTSD2, which is predicted as a methyltransferase gene. Further studies showed that the germination process of this mutant is not as so sensitive as that in wildtype to exogenous ABA, and pectin level rises in stem. These results make an excellent entry point to explore the regulation pathway of ABA, methyltransferase and pectin on seed germination. Basing on these results, techniques of prokaryotic expression and enzymatic assays, genetic transformation, immune labeling, metabolite detection and analysis, yeast one-hybrid et al will be employed to explore import events such as basic characteristics of OsTSD2 gene expression and action of its product，how OsTSD2 gene affects pectin components and methyl level during seed germination, relationship of ABA and OsTSD2 gene function, the OsTSD2 gene's upstream transcript factors screening and identification. Eventually the regulation pathway of ABA, methyltransferase and pectin on seed germination will be elucidated and role of this rice gene OsTSD2 on seed germination will be clarified.

种子萌发是种子植物生活史中的关键阶段，又是许多重要农作物生产的起点，对其过程机制研究具有重要的理论和实践意义。为研究水稻种子萌发机制，申请者利用T-DNA插入突变体库，筛选到一个萌发延迟的突变体。经侧翼分离和鉴定，其突变座位为甲基转移酶基因OsTSD2。进一步研究发现，其萌发过程对外源ABA敏感度下降、茎秆中果胶含量上升，这为研究ABA、甲基转移酶和果胶对种子萌发的调控途径提供了极好的切入点。拟在此基础上，利用原核表达、酶活测定、遗传转化、免疫标记、代谢产物检测与分析、酵母单杂交等技术，研究OsTSD2基因及其产物作用的基本特征、OsTSD2基因对萌发种子中果胶成分和甲基化水平的影响、ABA信号和OsTSD2基因功能的上下游关系、OsTSD2基因上游转录因子的筛选与鉴定，最终揭示ABA-甲基转移酶OsTSD2-果胶对种子萌发的调控途径，阐明水稻基因OsTSD2对种子萌发的影响机制。

果胶的合成和修饰对植物发育来说是非常重要的，虽然其具体的调控机理仍然不太清楚。本研究对水稻中的甲基转移酶基因OsTSD2进行了功能鉴定。首先获得了该基因的三个等位突变体，开展了突变体中与种子相关表型的系统分析。从结实率、粒型、颖壳颜色、萌发速率、果胶甲酯化水平等多个方面显示了OsTSD2基因的调控作用。为进一步揭示ABA—甲基转移酶基因OsTSD2—果胶三者是如何协作来调控水稻发育，我们利用了一个更为理想的研究系统即根揭示了果胶合成与修饰对细胞黏附性的影响。OsTSD2基因三个独立的等位突变体都表现出根不同区域的发育异常，这些异常包括异常的细胞粘附性、分生区裂生型通气组织的形成、伸长区细胞伸长的抑制和成熟区侧根密度的增加。利用免疫荧光标记（LM19）和钌红染色结果显示Ostsd2 突变体根中的非甲酯化HG增加。对细胞壁果胶多糖的生化分析则显示Ostsd2 突变体根中的单糖成分和糖醛酸含量都降低。除此之外，Ostsd2 突变体根中的ABA含量增加，ABA和IAA对细胞粘附性分别表现出抑制和促进的效果。这些结果暗示OsTSD2基因通过调控果胶的合成及修饰进而影响水稻根的发育。基于果胶对细胞伸长及分裂调控的区域化特点，该研究提出了一个模型，阐述了OsTSD2基因、果胶甲酯化和水稻发育之间的关系。