非蛋白巯基介导马蔺Pb向地上部长距离运输作用及机制研究

Phytoremediation of lead(Pb) contaminated soils by use of the Pb hyperaccumulators is considered to be the most promising ecological approaches. Iris lactea var. chinensis is an excellent ecological vegetation plant with the advantages of Pb hyperaccumulation and resistance ability. But, Pb is mainly distributed in the shoot. If we can take an effective means to increase Pb transport efficiency to shoot will improve phytoremediation effects of Iris lactea var. chinensis on Pb polluted soils. The non-protein thiols play important roles in heavy metal accumulation and long-distance root to shoot transport. Our preliminary studies have showed that Pb cuold induce high content of different non-protein thiols in Iris lactea var. chinensis and had a significant promoting role in Pb transport to the shoot, but which non-protein thiol is the key factor and the transport mechanism of the non-protein thiols in Iris lactea var. chinensis are unclear up to date. Thus, our study project will firstly clear the role of non-protein thiols during the Pb long-distance transport in Iris lactea var. chinensis through studying the effects of Pb and exogenous L-buthionine sulfoximine (BSO), Glutathione (GSH) and Cysteine (Cys) on the non-protein thiols contents,Pb contents and Pb forms. Futhermore, we will study the effect of exogenous BSO, GSH and Cys on the relations between Pb compartmentalized distribution,and the correlation of non-protein thiols synthase gene expression and Pb transfer efficiency in Iris lactea var. chinensis by use of isotopic tracer and RT-PCR techniques, and clarify the physiological and molecular mechanisms of non-protein thiols regulating Pb long distance root to shoot transport of Iris lactea var. chinensis. Moreover, It will provide a new target for creating and fostering high efficient transit-type hyperaccumulators and accelerating phytoremediation of Pb-contaminated soil in the future.

利用铅超积累植物修复铅污染土壤是最有前景的生态治理途径。马蔺是一种超强富集和耐受铅且具备多种优异抗性的生态植被材料，但其体内铅多积累于根系，若能采取有效手段增加铅向地上部转运将更加有利于马蔺的修复应用。非蛋白巯基在植物重金属积累和向地上部长距离运输中起重要作用，我们前期研究表明铅诱导马蔺产生大量非蛋白巯基，且对铅向地上部转运具有明显促进作用，但何种非蛋白巯基起关键作用及作用机制尚不清楚。为此，申请者拟首先研究铅胁迫以及添加外源BSO、GSH等对马蔺根系、地上部及木质部非蛋白巯基含量、铅含量和铅形态影响，明确何种非蛋白巯基在马蔺铅长距离运输中起关键作用；然后利用同位素示踪和QRT-PCR技术研究非蛋白巯基对马蔺铅区室化和铅转运效率的影响以及非蛋白巯基合成酶基因表达与铅转运效率相关性，阐明非蛋白巯基介导马蔺铅向地上部长距离运输机制，为创建培育铅高效转运型新种质加快植物修复铅污染土壤提供靶点。

马蔺是一种超强富集和耐受铅且具备多种优异抗性的生态植被材料，但其体内铅多积累于根系，若能采取有效手段增加铅向地上部转运将更加有利于马蔺的修复应用。非蛋白巯基在植物重金属积累和向地上部长距离运输中起重要作用，我们前期研究表明铅诱导马蔺产生大量非蛋白巯基，且对铅向地上部转运具有明显促进作用，但何种非蛋白巯基起关键作用及作用机制尚不清楚。因此，该项目主要开展了：1）不同浓度Pb胁迫下马蔺根系和地上部不同非蛋白巯基含量与Pb含量的相关性研究；2）外源非蛋白巯基GSH、Cys对马蔺 Pb 向地上部长距离运输作用的影响研究；3）外源GSH、BSO（GSH合成抑制剂）对马蔺 Pb积累和生长的影响研究；4）外源 GSH和BSO 对马蔺铅吸收动态及组织细胞分布的影响研究；5）非蛋白巯基合成酶重要相关基因γ-ECS、GS、PCS的表达与Pb积累转运的关系。结果表明：马蔺地上部和根系Pb含量与PCs和GSH含量在不同胁迫时间均保持显著的正相关，Pb-GSH和Pb-PCs是Pb在马蔺体内的主要非蛋白巯基肽结合形态，PCs和GSH诱导合成量的大幅增加是缓解马蔺体内Pb毒害的重要途径之一。马蔺根尖Pb超微定位结果表明，绝大部分Pb沉积在根尖细胞质外体细胞间隙中，其次为细胞壁，添加外源GSH使细胞间隙Pb急剧增多，说明添加外源GSH后细胞间隙和细胞壁仍是马蔺Pb积累的主要部位。Pb胁迫下添加外源GSH和BSO马蔺短时间Pb外流动力学实验也表明短时间（0-4 h）Pb 胁迫下马蔺根系吸收的 Pb可能主要积累于马蔺表皮细胞壁中，Pb+GSH 处理下马蔺根细胞解析出的 Pb 含量均高于单 Pb 胁迫和 Pb 和 BSO 处理下相应部位的 Pb 含量，说明 短时间内 GSH 具有促进马蔺 Pb 吸收和转运的功能。Pb胁迫下添加外源GSH 4-24 h GS基因相对表达量均高于单Pb胁迫下GS基因表达量，但处理4 h时GS基因表达上调极为显著，地上部和和根系相对表达量分别是单Pb胁迫下GS基因相对表达量的1.82和4.04倍，其余时间与单Pb胁迫下GS基因表达量差异不明显。而γ-ECS和PCS基因表达上调水平低于GS基因表达上调水平。因此，Pb胁迫下添加外源GSH后马蔺根系和地上部GS基因表达的显著上调以及PCs和GSH诱导合成可能是Pb积累和转运增加的重要机制。是下一步提高马蔺Pb修复效应的有效靶点。