水稻液泡型H+-ATPase A1亚基控制种子休眠的生理机制与分子调控网络

Seed dormancy is an important physiological stage for plant life cycle and a double-edged sword for crop production. Loss or reduced seed dormancy is the key aspect directly related to pre-harvest sprouting which leads to loss of crop grain weight and quality. However, strong dormant seeds germinate unevenly or not at all，which will affect the progress of crop production. Thus, it is very important to study molecular and physiological mechanism of seed dormancy and develop the moderately dormant rice varieties for protection of food security in China. In our previous studies, the vha-A1 mutant was identified by 60Co γ-radiation treatment of indica restore N142, which showed seed dormancy attributable to physical (defect of micropyle structure) and physiological (ABA sensitivity) factors. Genetic analysis indicated that vha-A1 was controlled by a recessive nuclear gene, which was finely mapped to a 42 kb interval between two markers RM20540 and RM20542 on long arm of chromosome 6. Sequence analyses revealed a 1-bp deletion of C in the open reading frame (ORF) region of the vha-A1 gene (LOC_Os06g45120), which is predicted to encode vacuoler H+-ATPase subunit A1. The cloned gene was confirmed by genetic complementary test. In this study, in order to confirm VHA-A1 is very important for seed dormancy, we will investigate the relationship among the vacuolar H+-ATPase subunit A1 on rice seed development, especially the development of micropyle structure; Physiology of metabolism and molecular regulation network of VHA-A1 gene on rice seed dormancy; The phosphorylation sites of VHA-A1 and the kinase for its phosphorylation. Based on the results of above, the physiological mechanism and molecular regulation network of VHA-A1 on seed dormancy will be involved in rice.

种子休眠性是植物生命活动的一个重要生理阶段。对农业生产来说，种子休眠是一把双刃剑。种子休眠性减弱甚至丧失将造成作物穗发芽并降低其产量与品质；而种子休眠性过强则会导致种子不能萌发或者出苗参差不齐，影响生产进程。因此，深入研究种子休眠的分子生理机理，培育适度休眠的水稻品种，对保障我国粮食安全具有十分重要的意义。在前期的研究中，申请人辐射诱变获得水稻种子休眠突变体vha-A1，其主要特征种子萌发孔存在缺陷，对O2及ABA敏感。利用图位克隆技术证实VHA-A1就是控制突变体vha-A1种子休眠特性的基因，该基因编码液泡H+-ATPase A1亚基。本项目将在前期的研究基础上，研究VHA-A1调控水稻种子生长发育的细胞生物学特点，VHA-A1调控水稻种子休眠的代谢生理与分子调控网络，以及VHA-A1的磷酸化位点及其激酶，从细胞、生理和分子角度揭示VHA-A1控制水稻种子休眠的生理机理及分子调控网络。

种子休眠性是植物生命活动的一个重要生理阶段。对农业生产来说，种子休眠是一把双刃剑。种子休眠性减弱甚至丧失将造成作物穗发芽并降低其产量与品质；而种子休眠性过强则会导致种子不能萌发或者出苗参差不齐，影响生产进程。因此，深入研究种子休眠的分子生理机理，培育适度休眠的水稻品种，对保障我国粮食安全具有十分重要的意义。本项目利用辐射诱变获得的水稻种子休眠突变体vha-A1开展的主要研究内容包括：1）突变体vha-A1种子的细胞结构特征；2）突变体vha-A1种子休眠的激素及ROS生理；3）不同处理条件下突变体vha-A1萌发种子的差异表达谱及其共表达基因分析；4）VHA-A1共表达基因OsHXK9调控水稻种子萌发的验证研究；5）实用水稻穗萌抑制剂的开发研究。结果表明，突变体vha-A1种子萌发孔明显存在结构缺陷，致使种子胚中的H2O2累积不足而引起种子萌发率降低。不同浓度的外源H2O2处理突变体种子则可以使其种子萌发恢复正常，从而进一步证实H2O2是影响突变体种子萌发率低的关键生理因素。进一步深入研究H2O2累积不足的生理原因，结果发现突变体萌发种子的SOD酶活显著低于野生型对照。不同条件下萌发种子的差异表达谱表明有很多激酶基因、ROS调节基因以及细胞分裂相关基因参与突变体种子萌发。进一步利用基因编辑激素敲除其中一个VHA-A1的共表达己糖激酶基因OsHXK9，结果发现敲除突变体oshxk9的种子萌发率显著降低，而且突变体种子中的ROS含量也显著低于对照，同时外源H2O2也可以恢复突变体种子萌发率，证实ROS介导了OsHXK9基因调控种子萌发。为了降低水稻穗萌发率，基于己糖激酶调控种子萌发的结果，研发己糖激酶抑制剂和铜的混合穗萌抑制剂。上述研究结果将为培育合理休眠的水稻种质以及降低水稻穗萌奠定基础。