基于iTRAQ技术的种子脱水耐性蛋白质组学研究

Desiccation tolerance is the basis for the long-term storage of seeds, but only orthodox seeds can aquire this trait, and recalcitrant seeds are desiccation sensitivity. Until now, the molecular mechanism of seed desiccation tolerance is still unclear. With recalcitrant coca seeds (Theobroma caca) and orthodox pea seeds (Pisum sativum) as experimental materials, this project will firstly establish a comparable system for desiccation tolerance research using a chemical method, and then use the isobaric tags for relative and absolute quantification (iTRAQ) technique as the main proteomic methods in combination with the two-dimensional gel electrophoresis technique and real-time PCR to investigate (1) the proteomic difference among seeds with different desiccation tolerance; (2) the proteomic difference during drying of seeds with different desiccation tolerance; (3) the gene expression of candidate proteins related to desiccation tolerance during CaCl2 and MV treatment and desiccation; and (4) the time courses of expression of key genes involved in the candidate metabolic pathway related to desiccation tolerance, which was seeked from the KEGG pathway database. It aims to clarify systemically the mechanism of seed desiccation tolerance from the proteomics and reveal the key proteins/genes or metabolic pathway related to desiccation tolerance. These results will support the new knowledge and technology for long-term storage of recalcitrant seeds.

脱水耐性是种子长期保存的前提，但只有正常性种子具有脱水耐性，而顽拗性种子对脱水敏感。迄今，种子脱水耐性的分子机理仍然不清楚。本项目以顽拗性可可和正常性豌豆种子为材料，利用化学方法构建可比的脱水耐性研究体系，以同位素标记相对和绝对定量 (iTRAQ) 蛋白质组学研究技术为核心方法，结合双向电泳以及实时荧光定量PCR技术，研究 (1) 不同脱水耐性种子之间的蛋白质组差异；(2) 不同脱水耐性种子脱水过程中蛋白质组的变化差异；(3) 脱水耐性相关的候选关键蛋白对应基因的时间表达特征； (4) 并参考KEGG途径数据库发掘与种子脱水耐性相关的关键代谢途径，研究代谢通路中的一些关键基因时间表达特征和代谢物含量变化，旨在从蛋白质组学水平系统地阐明与种子脱水耐性相关的机制，并发掘脱水耐性相关的关键蛋白/基因以及代谢途径。研究结果将为顽拗性种子的长期保存提供新的知识与技术。