苎麻BnPCS1调控镉胁迫应答基因的分子机制

Ramie (Boehmeria nivea), also named “China grass”, is an important source of cellulose-rich natural fibres which has rapid growth, higher biomass and rich genotypes. It is significative to renovate heavy metals from contaminated soil with ramie due to its high accumulation and high economic value. For the Cd contaminated land, culturing ramie is a candidate measure for safely reuse of the land, since ramie could minimizes the potential hazard of bringing toxic metals into food chains. The tissue-specific analysis showed that the BnPCS1 was expressed in root, stem, shoot tip, young and mature leaves with different expression level. BnPCS1 responsed to Cd stress. We obtained transgenic Arabidopsis thaliana of BnPCS1 gene whose Cd tolerance was higher than that of WT Arabidopsis thaliana. Additionally, one positive interacted proteins was obtained from a cDNA library of ramie. In this study, we would like to investigate conforming that BnPCS2 interact with BnPCS1; exploring BnPCS1 directly target genes and influence factors; the mechanism of BnPCS1 and BnPCS2 involved in Cd stress response by using genetics, cellular biology, molecular biology, and biochemistry means, and also explore their possible application in plant Cd tolerance improvement.

苎麻是原位修复重金属污染耕地的模式植物之一。研究苎麻耐镉分子调控机理将为充分发挥苎麻生态修复功能、调控植物镉富集能力提供理论基础。目前植物耐镉的分子调控机理尚不十分清楚，本项目组前期克隆了苎麻植物螯合肽合成酶基因BnPCS1，BnPCS1在苎麻成熟叶中表达量最高，受Cd诱导上调表达；BnPCS1过表达增强了拟南芥耐Cd能力和Cd积累量，符合BnPCS1在作物耐镉过程起重要作用这一假设。此外，我们利用酵母双杂系统筛选苎麻全生育期的 cDNA 文库，筛选了1个阳性互作蛋白。本申请将在此研究的基础上，结合遗传学、分子生物学和生理生化等技术手段进一步解析BnPCS1互作蛋白的功能，明确BnPCS1及其互作蛋白的协调互作关系；挖掘BnPCS1基因组水平的靶基因，从分子水平绘制BnPCS1调控植物镉胁迫应答基因的调控网络。预期研究结果对于植物修复的应用以及培育超抗或超积累新品种具有十分重要的意义。

苎麻是原位修复重金属污染耕地的模式植物之一。植物螯合肽合成酶基因BnPCS1参与植物响应镉胁迫，目前，BnPCS1功能研究尤其是调控植物镉胁迫应答基因的研究还比较少。在本项目的资助下，我们利用酵母双杂交鉴定了BnPCS1互作蛋白BnCLPB3，分别创建了BnPCS1和BnCLPB3基因超表达拟南芥转基因材料，获得了各自相应的转基因纯和株系，重点研究了BnPCS1、BnCLPB3的生物学功能及其调控机制。研究发现，BnPCS1和BnCLPB3响应镉胁迫，受镉诱导显著上升表达；BnCLPB3还响应PEG胁迫，亚细胞定位显示BnCLPB3位于细胞膜。进一步，利用转录组测序（RNA-Seq）、real-time PCR 检测 BnPCS1 过表达转基因拟南芥在基因表达水平的差异，发掘新基因1,135个，其中358个得到功能注释，筛选出14,881个差异表达基因。初步确定了 BnPCS1 调控的下游基因。靶向基因大多参与生物学过程，且显著富集到重金属胁迫响应、重金属转运等通路。另外，本研究发现BnPCS1诱导NTP的生物合成和消耗信号路径，同时参与氧化应激反应维持细胞内的活性氧平衡。综上所述，本研究结果对于植物修复的应用以及培育超抗或超积累新品种具有十分重要的意义。