利用转录组与蛋白组关联分析揭示花生镉吸收转运的分子机制

High cadmium (Cd) concentration in peanut kernel has become one of the principal factors that restrict the export of peanut. How to control Cd pollution in the peanut kernel is the most important problem in peanut safe production. Currently, the physiological mechanism of Cd accumulation in peanut kernel has been extensively studied, however, its molecular mechanism is not yet fully understood. The present project aims to investigate the responses of transcriptomic and proteomic profiling to Cd exposure and elucidate the molecular mechanism involved in the uptake and translocation of Cd in peanut. Transcriptomics and proteomics in roots, stems, leaves and seeds (ovules) of two contrasting peanut cultivars in seed Cd accumulation will be analyzed by using RNA-seq and iTRAQ (isobaric tags for relative and absolute quantitation) technology, under low Cd and control conditions. On the basis of this, integrative transcriptomic and proteomic analysis will be conducted to identify the key genes involved in the uptake and translocation of Cd in peanut. Thereafter, RACE (rapid-amplification of cDNA ends) technology will be utilized to clone the key genes involved in Cd uptake and translocation, and the subcellular localization will also be performed. The expression patterns of key genes in different tissues will be analyzed by using qRT-PCR. The implementation of the project is expectedly to build a molecular regulatory networks for Cd uptake and translocation in peanut plants, elucidate the the molecular mechanism of cultivar differences in seed Cd accumulation in peanuts, and provide a theoretical basis for effective control of peanut seed Cd pollution via screening and breeding of Cd pollution-safe cultivars by using molecular genetics methods.

花生籽粒镉含量偏高已成为制约我国花生出口的主要因素之一。如何控制花生籽粒镉污染，是当前花生栽培和食品安全面临的首要问题。目前，我们已基本搞清了花生籽粒镉积累品种间差异的生理机制，但对其分子机制尚不清楚。本项目拟以高/低镉积累花生品种为研究对象，采用RNA-Seq转录组测序技术和iTRAQ 蛋白组学分析技术，通过对镉诱导下花生根、茎、叶、种子(胚珠)的基因表达谱和蛋白质谱的品种间差异的比较和关联分析，挖掘不同组织中参与镉吸收转运的关键基因。采用RACE技术克隆参与镉吸收转运的未知关键基因，并进行亚细胞定位。采用qRT-PCR技术，分析不同组织中镉吸收转运关键基因的表达模式，探讨关键基因对花生镉代谢的调控机理。本项目的实施，有望构建花生镉吸收转运的分子调控网络，阐明花生种子镉积累品种间差异的分子机制，为通过分子遗传学方法筛选和培育镉污染预防花生品种、有效控制花生籽实镉污染提供理论依据。

本项目以丰花1号（低镉品种）和四粒红（高镉品种）为研究对象，采用RNA-Seq转录组测序技术和iTRAQ 蛋白组学分析技术，对镉诱导下花生根的基因表达谱和蛋白质谱的品种间差异的比较和关联分析，挖掘根中参与镉吸收转运的关键基因，明确了影响镉吸收转运的关键环节及其分子调控网络。比较转录组研究表明，IRT1、PDR12、YSL7、ZIP1、FRD3、ZIP11、MTP4、ABCC4和ABCC15等转运蛋白基因在丰花1号中的表达高于在四粒红，可能是造成花生植株Cd积累差异的原因。细胞壁相关蛋白（PEs、 PGIPs、 GTs、XYT12、 CYP450s、 LACs、 4CL2、C4H和CASP5）基因在丰花1号中的高表达可能有助于降低Cd向木质部的移动并向地上部分迁移。比较蛋白质组研究表明，一些与细胞壁修饰有关的蛋白质，如PEs、PE8B、PGIP2、XTH31、PODs、LACs和CCoA-OMT，在丰花1号中的表达丰度高于四粒红，说明其不仅具有高的防止果胶分解和去甲基酯化的能力，还可能通过增加木质素含量，从而阻止Cd进入根细胞，从而减少Cd由地下部分向地上部分转运。在上述研究的基础上，成功克隆了AhNRAMP1、AhIRT1、AhZIP1和AhZIP5等参与花生镉吸收转运的关键基因，并对这些基因在花生镉吸收转运过程的功能进行了解析。这些结果可以为花生品种间Cd积累差异的分子机制提供新的认识，对花生低Cd品种的选育具有重要意义。发表论文8篇，其中SCI检索论文7篇。培养在读硕士生1名。