重金属跨膜吸收的通道转移机制及电生理特征

In order to assure safety of food products of plant origin and to amend the soils polluted by heavy metals, it is necessary to track relevant functional genes so as to develop the required plants with specific functions. The primary step towards this target should be to understand the physiological characteristics (including the pathways for transmembrane transport, factors affecting the transport process and changes of relevant physiological parameters) of heavy metals during their entrance into plant cells. Real uptake of heavy metals is theoretically confined to the physiological process that the heavy metals enter the cytoplasm across membrane, rather than to the adsorption on root surface and chemical deposition in water free space of the apoplast. However, the mechanisms pertaining to the transmembrane transport of heavy metals have not been so clear yet, and some previous reports are even contradictory. In this project, different genotypic plants with various stress-tolerant strategies (Strategy I and Strategy II) and with differed ability in heavy metals accumulation are to be used as materials to respectively investigate the transmembrane transport process of three typical heavy metals (Cd, Cr and Cu), using biochemical methods including the use of metabolic and channel inhibitors and in vitro assessment of cell enzymes and electro-physiological techniques (namely micro electrodes) and by means of kinetics of nutrient uptake and collection of xylem sap. Among three heavy metals involved in the study, Cd usually exists in the form of Cd2+ and is more responsible for soil pollution; Cr is characterized with its various valence states and in soil it usually exists in the form of Cr3+ or CrO42-; while the representativeness of Cu indwells in that it belongs to heavy metal and simultaneously serves as an essential element which is the constituents of many enzymes responsible for important physiological reactions in plant body. The project targets to answer following scientific questions: From what part of the root the heavy metals enter the plant body and then transport to overground part along the xylem? In what form the heavy metals are absorbed by cells (in ion form or in complex state)? For what kind of channels on the membrane the heavy metals compete with other cations during their transport across the membrane? And what environmental or metabolic factors can affect the transmembrane transport of the heavy metals? The specialized physiological characters of these heavy metals displayed during their transport process across the cell membrane are expected to be identified through this study, which can provide references for future investigation on functional genes involved in regulation process so as to fundamentally support the development of plant materials with high potentials in accumulating or excluding heavy metals by means of molecular biological approaches.

为提高植物食品的安全性和利用植物修复重金属污染土壤，需要追踪有关的功能基因以培育具有特殊功能的所需植物。为此首先应寻找重金属在吸收进入细胞过程中所表现出的生理特征，但迄今对其跨膜转移机制的了解还欠清晰。本研究拟采用具有不同抗逆机理（机理I和机理II）和不同重金属积累能力的植物基因型作为实验材料，利用代谢抑制剂和通道抑制剂以及离体细胞酶测定等生物化学方法，离子选择性微电极等电生理测定技术，配合营养吸收动力学和木质部液汁收集等研究手段，对三种典型重金属（Cd、Cr、Cu）的跨膜转移过程进行研究，搞清它们是以离子态还是络合态被植物吸收，在跨膜进入细胞时与其他阳离子竞争位于细胞膜上的哪些离子通道，哪些环境或代谢因子影响它们的跨膜转移等问题。通过研究，鉴别出它们在跨膜转移过程中所表现出的特有生理特征，为进一步追踪参与调节的功能基因和在此基础上采用分子生物学手段挖掘植物排斥或积累重金属的潜能提供参考。

植物对重金属的吸收涉及到食品安全。对不同植物根系吸收重金属的机制研究有助于发现不同植物在吸收和转移重金属的过程中的特有生理特征，可据此进一步追踪参与调节的功能基因，为采用分子生物学手段挖掘植物排斥或积累重金属的潜能提供参考。本项目主要采用常规营养吸收培养方法、电生理方法、非损伤微电极测定方法以及电镜等，围绕不同植物或品种对重金属的吸收、积累特点、不同根系部位对重金属吸收的贡献、重金属吸收过程中根表皮细胞膜电位的变化及其与质子泵活性的关系等进行研究。所获得的重要结果、关键数据和科学意义可归纳如下：（1）豆科植物单位根表面积对重金属的吸收速率大于禾本科类植物，在根尖部位差异尤其显著。（2）随距根尖距离的增加，植物的单位根表面积重金属内流速率降低。但对水稻而言，其成熟区独有的表皮厚壁纤维细胞阻挡了重金属的吸收。（3）不同品种水稻单位根表面的有效通道数不同。（4）采用不同的根系参数计算出的吸收速率可能得到不同的结果。在利用这些参数指导遗传育种时，不能仅关注某一参数。（5）同种重金属的不同形态离子在跨膜转移中可能通过不同的离子通道。（6）重金属的络合形态对植物吸收存在影响。络合剂的络合能力越强，植物对该重金属的吸收越难。（7）对重金属的吸收会瞬时导致植物根细胞膜的去极化，但随时间延长，逐渐向原始态恢复，其恢复速度与恢复程度应不同品种而异。（8）重金属吸收导致的细胞膜去极化进一步刺激了H+-ATPase的活性，使其泵氢能力增强，这种反应的强弱应品种不同而异。（9）发现了细胞膜电位变化可能具有信号传导作用，这对探讨植物体内的长距离信号传导系统的研究具有启发。（10）重金属之间的吸收有的表现拮抗，有的表现促进，有的相互影响不大；而重金属与养分离子吸收间基本表现为拮抗关系。（11）无机阴离子的种类对植物吸收重金属也有一定的影响。（12）研制出了一种用于测定铜离子的非损伤微电极制作方法。

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