Pb超富集植物金丝草对Pb的解毒机理及耐性策略

Pb hyperaccumulator plant—Pogonatherum crinitum was found by our research teem previous for the first time in China, which can grow normally in serious Pb contaminated environment. But currently it is still not clear of the tolerance strategies and detoxification mechanism of Pogonatherum crinitum to Pb stress. Morphological and physiological detoxification strategies were researched by atomic absorption spectrophotometer，reverse high performance liquid chromatography (HPLC), ion exchange chromatography, electron microscopy, PCR high purity identification and TUNE. Differentiation law of morphophysiology of leaf and root was researched in different Pb stress concentration of Pogonatherum crinitum. Compartmentalization position of Pb complexation in leaf organiation was scanned. And differences of intracellular scalariform DNA, organic acid, homocysteine protease actiity, cysteine protease activity, metallothionein, scavenge reactive oxygen species were measured. Relationship between tolerance properties to Pb stress and activating programmed cell death of Pogonatherum crinitum was revealed. The differences of morphological and physiological characteristics under different pulse conditions of Pogonatherum crinitum were conducted and sensitive and accuracy of detoxification mechanism was revealed. Finally, tolerance strategies and detoxification mechanism of Pogonatherum crinitum to Pb stress was revealed, which had important theoretical and practical significance to discover Pb enrichment and speeding up of soil repair to Pb pollution distribution.

课题组前期研究在国内首次发现了Pb超富集植物金丝草（Pogonatherum crinitum），可在高浓度Pb环境中正常生长，但其对高浓度Pb胁迫的解毒机制尚不清楚。本项目采用自主设计的异质及脉冲Pb胁迫试验装置，利用根系分析仪、离子交换层析、PCR高纯鉴定、反相高效液相色谱和电镜等手段，研究金丝草根系及叶片对Pb胁迫的形态学和生理学解毒策略，比较不同Pb胁迫条件下金丝草形态学指标的响应差异，分析金丝草叶片组织内Pb络合区室化的部位；定量测定金丝草胞内梯状DNA、有机酸、半胱氨酸蛋白酶活性、金属硫蛋白、活性氧清除系统等指标，探讨金丝草对Pb胁迫的生理学响应及其与激发其组织细胞程序性死亡的关系。分析金丝草在高Pb环境中形态和生理学解毒过程的启动机制，最终揭示金丝草对Pb胁迫的解毒机理及耐性策略。这对于发掘Pb超富集植物对Pb的富集潜力、加快Pb污染土壤的修复具有重理论和现实意义。

金丝草是课题组前期研究筛选出的一种Pb超富集植物，其可在高浓度Pb胁迫环境下正常生长，对Pb具有强的耐性和富集能力，为揭示其对Pb的解毒机理。本项目采用异质和脉冲Pb胁迫试验，通过测定不同Pb胁迫条件下金丝草的根系叶片形态、Pb的亚细胞分布等形态学指标，不同组织的荧光学特性、半胱氨酸蛋白酶活性、金属硫蛋白、活性氧清除系统、疏基（T-SH）、基脯氨酸（PRO）、Caspase3活力等生理学指标，以及根际土壤酶和微生物等指标，比较不同Pb胁迫条件下金丝草形态和生理指标的差异，发现了金丝草对Pb胁迫的解毒机理和耐性策略。研究结果表明，金丝草在遭遇土壤Pb胁迫后，通过根系的快速延伸避让Pb胁迫环境，在无法避让时通过增大根直径和表面积适应Pb胁迫环境；提高光的利用效率，增强SOD和POD抗氧化酶活性，清除活性氧自由基，细胞壁固定，机酸络合在解毒Pb中起着重要的作用，但未发现有脉冲启动机理；总疏基（T-SH）、基脯氨酸（PRO）在胁迫中期发挥了解毒作用，但在胁迫后期作用不明显，Caspase3活力先增大后减小，说明金丝草对Pb的解毒机理存在阶段性的响应机制。金丝草对Pb的耐性和解毒与加快细胞程序性死亡有一定关系。本项目发表了15篇标注国家自然科学基金资助的论文，其中SCI收录论文6篇，投稿录用论文4篇，获授权专利1项，培养已毕业1位博士和2位硕士研究生。这些研究结果丰富了植物抗逆胁迫的形态、生理学学科，为土壤重金属污染修复技术提供了一个新的突破口和途径，对从机理水平调控植物修复技术，加快重金属污染土壤的生态修复具有重要意义。