阴离子通道调控沙冬青气孔关闭机理研究

Drought is one major environmental stress that greatly affect plant growth and development all over the world. Guard cells form stomatal pores in the leaf epidermis, which enable plants to balance CO2 uptake for photosynthesis and water loss via transpiration. The highly-effective control of stomatal movement is one of the key mechanisms in response to drought stress. Ammopiptanthus mongolicus (Maxim), is the only evergreen broadleaf shrub grown long- term in the central Asian desert, the efficient closure of stomatal pore might be the key path for its survival. The opening or closure of stomatal pore is mainly determined by ion influx or efflux from guard cells by transporters or channels. Out-rectifying potassium channels and anion channels play key function in stomatal closure. Recently, we have reported that potassium efflux mediated by out-rectifying potassium channel in Ammopiptanthus is highly functional conserved with that in Arabidopsis, therefore, we presume that anion channel of guard cells in Ammopiptanthus may be the critical factor for stomatal closed fast and efficient to adapt to drought environment. Although, there are extensive studies on function and regulation mechanisms of SLAC1 gene in Arabidopsis and rice, it remained/s unclear in the basic electrophysiological features and functional conservation or specificity of SLAC1 channel. In this project, Ammopiptanthus, a kind of drought-tolerant plants, will be used as experimental materials. Firstly, we will try to clone the AmSLAC1 gene, in addition, the tissue specific expression features and sub-cellular localization of AmSLAC1 will be analyzed. Then, in one hand, by using TEVC and patch clamp technique, the kinetics features of AmSLAC1 channel (external anion concentrations, affinity constant for anions)，gating properties and voltage range allowing AmSLAC1 channel opening will be intensively studied; in the other hand, identification biological function of AmSLAC1 in vitro and figuring out its contribution to stomatal closure will be carry out via genetically modified and genetic methods. Finally, screening and identification some proteins in Ammopiptanthus which will interact with and regulate the activity of AmSLAC1 channel by yeast two-hybrid system. All of these results will be taken together to explain the regulation mechanism of Ammopiptanthus stomatal closure mediated by guard cells anion channel. These studies will provide a useful theoretical support and excellent genetic resources to improve plant drought resistance through molecular breeding.

干旱是影响植物生长发育最主要的环境因素之一。气孔作为植物体内水分散失的主要途径，其高效调控是植物适应水分胁迫的关键之一。研究发现，保卫细胞阴离子通道对气孔关闭起着非常重要的作用。目前，对其功能及调控研究多集中在非旱生植物如拟南芥等，而对典型旱生植物保卫细胞慢阴离子通道功能和调控机制尚未报道。本研究以极端干旱地区的典型灌木—沙冬青为研究材料，因其长期生长在极端干旱的环境中，对气孔高效关闭有着强烈需求，其保卫细胞慢阴离子通道对气孔关闭的作用是怎样的，是否形成旱生植物特有的功能调控机制，还有待研究。本研究拟克隆沙冬青慢阴离子通道基因并分析其表达定位特征。其次运用电生理和遗传学方法，研究该通道的功能；最后筛选沙冬青cDNA文库，搜寻体内能调控该通道的蛋白，探索沙冬青慢阴离子通道功能特征及对气孔关闭的调控机制，深入阐明旱生植物沙冬青适应干旱胁迫分子机理，为通过分子育种改良植物抗旱性提供优秀基因资源。

干旱是影响植物生长发育最主要的环境因素之一。气孔作为植物体内水分散失的主要途径，其高效调控是植物适应水分胁迫的关键之一。研究发现，保卫细胞阴离子通道对气孔关闭起着非常重要的作用。本研究以极端干旱地区的典型灌木——沙冬青为研究材料，因其长期生长在极端干旱的环境中，对气孔高效关闭有着强烈需求，通过探索沙冬青阴离子通道功能特征及其对气孔关闭的调控机制，对改善植物的抗旱能力，提高农作物产量，具有重要意义。.本文以蒙古沙冬青为实验材料，采用分子生物学、细胞生物学以及电生理学等研究技术手段，对慢阴离子通道AmSLAC1的生理功能及其作用机制进行了以下研究：1克隆AmSLAC1，对AmSLAC1的表达及亚细胞定位进行分析，明确其时空表达模式及其在细胞中的分布。表达分析结果显示AmSLAC1主要在沙冬青地上部表达，特别是保卫细胞中，此外该基因的表达受PEG及ABA所诱导，这与其参与了调控保卫细胞运动进而增强抗旱性的表型相一致。亚细胞定位结果显示其主要在细胞膜中表达。2通过异源表达，在模式植物拟南芥中构建了AmSLAC1/slac1-3缺失突变体互补株系和AmSLAC1过表达株系，通过对其干旱处理后的表型、叶片水分散失率、叶片气孔开度、光合效率等指标的观测和比较，表明AmSLAC1能够恢复slac1突变体对干旱胁迫的表型。3对AmSLAC1进行了蛋白互做分析，酵母双杂交实验和双分子荧光互补实验均显示，AmSLAC1能够与蛋白激酶AmOST1、AmCPK6相互作用。4对AmSLAC1进行了电生理功能分析，AmSLAC1需要与AmCPK6或AmOST1共同作用，才能行使转运Cl-的功能。另外，所检测的卵母细胞电流既有正向也有负向，表明AmSLAC1对Cl-转运是双向的，既能向细胞内也能向细胞外转运。