龙葵对镉超富集的差异蛋白表达特征及调控机理

Cd is one of the main pollutants in farmland soil in China. Constructing the technology of co-planting hyperaccumulator and low accumulation crop via the methods of intercropping or multiple-pant is undoubtedly of practical significance for the remediation of Cd contaminated farmland soil with huge area. Solanum nigrum L. was a newly discovered hyperaccumulator by applicant with higher potential of phytoremediation. Due to its too many priorities like wide distribution and large biomass, S. nigrum is an ideal remediation plant in the technology of abovementioned. This project is further deepening and expanding of the last completed project based on the new discoveries of “S. nigrum on Cd uptake showed a strong selectivity and metallothionein gene cloning and sequencing”. Furthermore, we have completed some research works on exploring hyperaccumulative mechanisms via plant physiology or biochemistry methods. Based on these results, hyperaccumulator S. nigrum (three different ecotypes) and non-hyperaccumulator (in the same family of S. nigrum but with low Cd accumulation) will be used to explore molecular mechanisms of hyperaccumulating Cd via the expression characteristics and regulation mechanism of differential proteins in this project. The main research contents including: a) expression and analysis of differential proteins in S. nigrum under the pollution of Cd or Cu with different concentration gradient; b) expression property and regulation of hyperaccumulator S. nigrum and non-hyperaccumulator (low accumulation to Cd); c) expression characteristics and regulation mechanism of three ecotypes of S. nigrum to Cd. Some modern scientific technologies such as iTRAQ, Western-blot and RPLC-Ms will be used in this project. All results will promote the application of phytoremediation in practice in the future.

镉是我国农田土壤主要污染物之一,构建超富集植物和低积累作物互作的“边修复边生产”技术对于污染面积巨大的镉农田污染土壤修复无疑具有实践意义。龙葵是申请人新近发现的镉超富集植物,生物量大，修复潜力很高。本项目在已完成的上一个基金项目所获成果“龙葵对镉吸收具有较强选择性以及金属硫蛋白克隆与测序”基础上，进一步深入和拓展，以不同生态型超富集植物龙葵和非超富集植物（与龙葵同科且对镉低积累）为试材，从差异蛋白角度揭示龙葵超富集镉的分子机理。主要研究内容有：1) 超富集植物龙葵对不同重金属污染响应的差异蛋白表达特征与分析；2) 超富集植物龙葵与非超富集植物差异蛋白表达特征与调控机理；3) 不同生态型龙葵对镉超富集差异蛋白的表达特征与调控机理。主要利用iTRAQ、Western-blot、RPLC-MS等蛋白组学现代分析技术，揭示龙葵超富集镉的差异蛋白表达特征与调控机理，为形成较成熟植物修复技术奠定基础。

龙葵(Solanum nigrum L.) 是申请人首次在沈阳地区发现的具有超富集植物全部特征的镉超富集植物。以不同生态型的沈阳龙葵和汉中龙葵为试材，开展“龙葵对镉超富集的差异蛋白表达特征及调控机理”研究具有重要实践意义。根据研究计划，已完成全部研究任务，完成了预期成果指标。发表SCI收录论文26篇。获陕西省高等学校科学技术一等奖一项。主要研究内容和结论如下： 1）超富集植物龙葵对不同重金属污染响应的差异蛋白表达特征与分析。采用水培培养投加不同浓度CdCl2，ZnSO4和CuSO4处理开展试验。结果表明，从代谢途径角度探究龙葵超积累Cd的机制，利用同位素标记相对和绝对定量(iTRAQ )技术，通过比较富集Cd， Zn和Cu积累相关的差异蛋白的比较,17个与Cd和Zn积累相关的共性特异性蛋白所反映的相关代谢途径包括吞噬体、氨酰- tRNA生物合成和碳代谢。差异蛋白所反映的与Cu不同的代谢途径可能是是龙葵超富集Cd的原因。进一步揭示了龙葵超积累Cd的分子机制。2）超富集植物龙葵与非超富集植物差异蛋白表达特征与调控机理。以超富集龙葵和低积累辽茄3号为研究对象，采用水培试验获得的超富集龙葵和低积累辽茄3号的关键蛋白表明，龙葵对镉的超富集可能是由于金属转运蛋白和转运相关ATP酶的高表达引起的，其中H+-ATP酶在生理和分子水平上对镉的转运起重要作用，而低积累茄子叶片和根中的H+-ATP酶下调表达，低浓度镉和锌胁迫使14-3-3蛋白和质膜H+-ATP酶上调表达，表明相关基因和蛋白质表达的变化与质膜质子泵活性的变化有关。 (3) 不同生态型龙葵对镉超富集差异蛋白的表达特征与调控机理。以沈阳龙葵和汉中龙葵为对象，在不同浓度镉胁迫下，沈阳龙葵和汉中龙葵与镉有15共性特异蛋白。高水平镉处理下，龙葵叶片叶绿体和淀粉合成酶Ⅰ蛋白下调表达。低水平镉胁迫对关键酶LOX、GR、cAPX、Cu/Zn SOD没有明显影响。