【作者】 张伟；

【导师】 武维华；

【作者基本信息】 中国农业大学 ， 植物学， 2005， 博士

【摘要】 胞质自由Ca²⁺是植物细胞普遍存在的第二信使,介导植物对体内外多种刺激产生相应反应。细胞质膜钙通道介导的Ca²⁺向胞质的转运对于胞质钙信号产生至关重要,因此,对植物细胞质膜钙通道进行研究并了解其调控机制,对于植物细胞信号转导网络研究具有重要意义。 本论文第一部分工作以蚕豆保卫细胞为实验材料,利用膜片钳等技术手段,对保卫细胞质膜钙通道进行记录、鉴定并对其调控因素进行研究,提出了渗透势在调控气孔运动过程中可能的作用机制。利用Ba²⁺作为通透性离子在全细胞水平记录到内向电流,逆转电位及抑制剂实验分析表明,该电流为Ba²⁺通过质膜钙通道内流形成。渗透势和微丝参与了全细胞钙通道电流的调控,胞外低渗和微丝解聚剂可以激活通道电流,且渗透势的作用是通过改变微丝结构进而调控通道的活性。单通道水平在蚕豆保卫细胞质膜上记录到张力激活型和电压依赖型两类钙通透性通道,其中张力激活型钙通道的激活依赖于质膜所受的张力,开放几率与张力大小成正比,张力激活型钙通道的开放还受跨膜电位调控,通道电流幅度与质膜超极化程度成正比,本实验条件下记录到的张力激活型钙通道电导为19.7 pS;蚕豆保卫细胞质膜上还存在有电压依赖型钙通道,其激活依赖于质膜超极化,单通道电导为14.3 pS。对这两类通道电流分别进行逆转电位分析、抑制剂鉴定、跨膜Ba²⁺浓度依赖性及Ca²⁺通透性等特性鉴定,表明实验中所记录的电流信号为跨质膜钙通透性通道的内向电流。同时发现,微丝参与了对张力激活型及电压依赖型钙通道的调控,微丝解聚后激活通道。进一步实验结果表明,渗透势对全细胞水平通道的激活作用至少部分通过张力激活型钙通透性通道来介导。利用激光共聚焦显微技术,测定渗透势、微丝解聚剂等因素对蚕豆保卫细胞原生质体胞质自由Ca²⁺浓度的影响,结果表明:胞外低渗、微丝解聚均可引起胞内自由Ca²⁺浓度的升高,且微丝解聚剂可在高渗下引起胞质Ca²⁺浓度升高,这些因素引起的Ca²⁺升高可以被胞外添加Ca²⁺通道抑制剂所抑制,说明胞内Ca²⁺浓度升高依赖于胞外Ca²⁺的流入。在胞内外都以Ca²⁺为通透离子的情况下,发现蚕豆保卫细胞质膜电压依赖型钙通道可以介导胞内K⁺外流,这在气孔保卫细胞对内外界刺激迅速作出反应中有重要意义。实验过程中在蚕豆保卫细胞质膜上还记录到一类高电导的钙通透性通道,单通道电导为正常通道电导的5倍,推测这类高电导通道的存在可能使某些保卫细胞对逆境信号作出更加快速的反应。 本论文另一部分工作对拟南芥可能钙通道基因进行克隆及功能鉴定。克隆到的拟南芥AtTPC1基因可以互补酵母电压门控钙通道CCH1的功能并恢复Ca²⁺吸收缺陷型cch1的性状。利用Promoter+GUS手段对AtTPC1基因进行了幼苗到开花期植株的组织表达定位,结果显示该基因在植株整体表达,但在各组织部位的表达强度不同。利用AtTPC1-GFP策略对瞬时表达的基因产物进行亚细胞定位,基因枪转化的洋葱表皮细胞显示,GFP荧光出现在膜结构区域。利用电压钳系统对体外转录后的AtTPC1基因cRNA产物进行电压钳记录,没有记录到电流,同时转录表达的KAT1基因cRNA产物作为对照可以记录到内向电流。利用AtTPC1基因功能缺失突变体及过表达突变体进行性状筛选,所选用的指标为高盐,低钾,高、低钙,高渗透势及干旱胁迫,但未发现与野生型植株间有明显差异。更多还原

【Abstract】 Cytosolic free Ca²⁺ ([Ca²⁺]_cyt) is a ubiquitous second messenger in plant cells. A number of external and internal stimuli trigger sustained or transient elevations of [Ca²⁺]_cyt and consequntly evoke downstream stimulus-specific responses. Generation of [Ca²⁺]_cyt signals depended on Ca²⁺ influx into the cytosol from the extracellular and intracellular Ca²⁺ stores through Ca²⁺ channels. Thus, Ca²⁺ channels in the plasma membrane play an essential role in Ca²⁺-mediated signaling process. However, the direct recordings and analysis of channel-facilitated Ca²⁺ influx are very limited and upstream regulation mechanisms of the Ca²⁺-channels in plant cells remain unclear so far.The first part of this dissertation work was focused on identification and functional characterization of the Ca²⁺-permeable channels in the plasma membrane of Vicia faba guard cells by application of the patch-clamp techniques. Under the whole-cell and single-channel configurations, inward Ba²⁺-currents were observed and identified. At the single-channel level, the stretch-activated （SA） inward channels were identified and characterized. The P_o （total open probability） of the SA channels depended on the stretch across the plasma membrane patches, and the current amplitude of the SA channels was voltage-dependent. The voltage-dependent or voltage-activated （VA） and stretch-independent inward channels were also recorded and analyzed. The analysis based on the inhibitory effects of Gd³⁺ and the reversal potentials demonstrated that both types of channels were Ca²⁺-permeable channels, which was further confirmed with the experiments using Ca²⁺ instead of Ba²⁺ as current carrying ions. The further experiments demonstrated that actin polymerization and depolymerization processes regulate the activities of both types of channels. Depolymerization of the actin filaments by application of cytochalasin D activated the channels （increased P_o of the channels）, and this activation was overcome when phalloidin was applied. In addition, the whole-cell Ca²⁺-currents were significantly regulated by changes in osmo-gradients across the plasma membrane. The inward Ca²⁺ currents were activated under external hypo-osmotic conditions, while the inward Ca²⁺ currents were inhibited under external hyper-osmotic conditions. More importantly, the osmo-regulation of the inward Ca²⁺-permeable channels is mediated by actin depolymerization and polymerization process. The further results support the notion that osmo-regulation of whole-cell Ca²⁺ currents were mainly contributed by the SA-type Ca²⁺-permeable channels.The second part of this work was cloning and preliminary functional characterization of AtTPC1, a putative Ca²⁺ channel in Arabidopsis thaliana （AGI code At4g03560）. AtTPCl has high homology with the Two Pore Channel in rat and its structure is very similar to the half of the structure of α-subunit of voltage-activated Ca²⁺ channels in animal cells. When hetero-expressed in yeast mutant cchl （a Ca24 uptake deficient mytant）, AtTPCl rescued the Ca²⁺ uptake activity of cch1. Because CCH1 encodes a homolog of the a-subunit of anima L-type Ca²⁺ channels, this result indicated that hetero-expressed AtTPCl is functional as a Ca²⁺ channel to facilitate Ca²⁺ influx.GUS staining results showed that AtTPCl is expressed in most of the organs of Arabidopsis such as roots, leaves and flowers. The result of transient expression of AtTPCl-GFP showed that AtTPCl may be located on the plasma membrane.In summary, this dissertation work investigated the Ca2+-permeable channels in the plasma membrane of Vicia faba guard cells. Two distinct types of Ca2+-permeable channels are identified and characterized. The SA-type Ca2+-permeable channels are osmo-regulated and the osmo-regulation the channels is mediated by actin polymerization/depolymerization process. A putative Ca2+-channel gene AtTPCl in Arabidopsis was cloned and function of AtTPCl for facilitating Ca2+-influx was demonstrated using yeast mutant.In addition, in collaboration with other members in the lab, regulation of the inward K+ channels in Arabidopsis pollen plasma membranes by Ca2+/CDPK signaling cascade was investigated. The results showed that the inward K+ currents were insensitive to [Ca2+]cyt in premature pollen protoplasts, whereas elevation of [Ca2+]cyt inhibited the inward K+ currents in mature pollen and pollen tube protoplasts. By analyzing the independent CDPK knockout mutants respectively, we found that the differences in [Ca2+]cyl sensitivity may result from differential expression of CDPK.更多还原