镉的亚细胞再分配差异调控油菜镉毒抗性的分子生理机制

Cadmium (Cd) in soils severely affects the production and quality of rapeseed in rice-rapeseed rotation regions of Southern China, and revealing the mechanisms underlying tolerance to Cd in rapeseed (Brassica napus L.) are an important prerequisite for genetic improvement of Cd resistance and breeding Cd hyper-accumulators. In this study, from a natural panel comprising 196 lines, we identified a Cd-tolerant cultivar Z11 and a Cd-sensitive cultivar W10, whose plant concentrations of Cd were not significantly, whereas Cd concentrations in the shoot vacuoles and root cell walls of Z11 were significantly higher than those of W10, respectively. We aim at fine isolation of subcellular components, further to determine the cell wall components, pectin concentrations and pectin methyl-esterification degrees, and study the differential metabolisms involving Cd-chelates in protoplasts and vacuoles between Z11 and W10 using HPLC-MS. We also will study the genetic variations and differential transcriptional responses of the genes related to pectin methylesterase (PME) in cell wall and ATP-binding cassette (ABC) transporters involving vacuolar sequestration of Cd through whole-genome re-sequencing and comparative RNA-seq, respectively. Through the construction of gene co-expression network and identification of their genetic variations, we ascertain the core members among the BnaPME and BnaABC family genes. Through the development of the transgenic rapeseed lines using the promoters of gene themselves and CRISPR/Cas9 systems, we aim at revealing the physiological metabolic bases and molecular detoxification mechanisms underlying tolerance to Cd regulated by differences in subcellular re-distribution of Cd in oilseed rape.

南方稻油轮作区土壤镉污染严重影响油菜的产量和品质，揭示油菜的耐镉机制是进行镉毒抗性的遗传改良、培育镉超累积品种的重要前提。本研究从196份油菜品系中筛选出耐镉品种Z11和镉敏感品种W10，二者植株镉浓度无显著差异，但Z11地上部液泡和根部细胞壁中的镉浓度显著高于W10。本项目拟通过亚细胞组分的精细分离，测定Z11和W10的细胞壁组分、果胶含量及其酯化度，利用HPLC-MS分析原生质体和液泡内小分子螯合物的代谢差异。分别利用全基因组重测序和比较转录组鉴定在Z11和W10中存在遗传变异和表达差异的果胶甲酯酶基因BnaPMEs和液泡膜转运基因BnaABCs。通过构建差异表达基因共表达网络，鉴定并克隆BnaPMEs和BnaABCs家族核心调控基因。通过构建基因自身启动子表达和CRISPR/Cas9敲除转基因油菜，揭示镉的亚细胞再分配差异调控油菜镉毒抗性的生理代谢基础及分子解毒机制。

镉（Cd）是生物毒性最强、污染程度最为严重的重金属之一。拟南芥和模式作物水稻中镉毒抗性的遗传和分子机制取得了较大的研究进展，但目前在油菜中仍缺乏从生理代谢、遗传基础和分子调控等方面对其镉毒抗性机制的系统深入解析。. 研究表明，油菜种间或种内的镉毒抗性存在显著的基因型差异；然而，目前关于油菜的镉毒抗性研究多集中在某一品种或者不同基因型的单一生理差异研究。研究并提高油菜的镉毒抗性是培育适合我国生态环境治理的镉超累积植物、实现植物修复镉等重金属污染土壤的重要前提。我们在本项目的资助下，在Journal of Experimental Botany（IF>5.0）发表了题为“A multiomics approach reveals the pivotal role of subcellular reallocation in determining rapeseed resistance to cadmium toxicity”（doi: 10.1093/jxb/erz295）的研究论文。我们从近200份油菜自然品系中鉴定到两个镉毒抗性差异基因型，首先利用ICP-MS进行离子组测定，发现二者器官水平镉吸收和转运并无差异，通过亚细胞组分的精细分离，但抗性品种的根部细胞壁和地上部液泡的镉显著高于敏感品种。通过体视显微镜、扫描和透射电镜发现油菜镉毒抗性品种和敏感品种在表皮毛、气孔等植株形态和亚细胞超微结构存在显著差异。进而通过全基因组重测序鉴定DNA变异、比较转录组挖掘差异表达基因以及UHPLC-MS辅助的代谢物差异分析，发现油菜镉毒抗性品种地上部镉螯合物含量和根部细胞壁果胶甲基酯化度显著高于镉毒敏感品种，并从分子水平揭示果胶甲酯酶PME和液泡ABC转 .运蛋白可能调控了油菜耐镉基因型差异的分子解毒机制。该研究将为油菜镉毒抗性的遗传改良和镉超累积植物的培育提供了优异的种质资源和优良的基因资源。