干旱胁迫下牡丹的转录组分析及相关基因克隆

Tree peony (Paeonia suffruticosa Andrews) is a well-known horticultural and medicinal plant in China. As the global climate change and water dificiency in the world, more researching work should be done on developing drought-tolerant varieties, improving culture technique,expanding suitable cultivation area. This research was firstly to explore the mixture and development time required during different drought stress by photosynthetic characteristic and physiological property analysis.Then,the RNA-Seq technique was employed to compare the gene expression profilings of drought-treated seedlings and wild type to screen the specific related genes with moutan drought-responsing characteristics. Subsequently, temporal and spatial expression regulations of the related genes were investigated by real-time quantitative PCR. Full length cDNAs of the related genes were cloned. Structural and functional analysis were carried out based on the full length cDNAs sequences. Plant expression vectors were constructed and functional analysis was conducted by Arabidopsis transformation. In this study, we aim to establish a normalized cDNA library prepared from leaflets of buding period moutan during drought treatment and obtain a substantial EST dataset using RNA sequencing technology, acquire some new independent intellectual property rights genes which have important function on moutan drought-responsing characteristics, ascertain the molecular mechanism of moutan drought-responsing characteristics. These data will provide a useful mean of gene discovery, genome characterization and global understand of drought response as well as novel insight into the expressed genes during drought induction in moutan. The results would have important theoretical and practical significance for mechanism study molecular breeding in moutan.

牡丹是中国的传统名花，具有重要的观赏和药用价值。应对全球气候干旱变化及水资源短缺的生态条件，进一步研究牡丹抗旱性提高的分子及生理机制，对培育抗旱新品种，提升栽培技术，扩大适生面积的意义重大。应用高通量测序技术，从转录水平在对比两个品种间基因表达谱差异的同时，分析未经任何处理的植株与经最长时间干旱胁迫仍能正常生长植株的基因表达谱，筛选出其响应干旱胁迫的特异候选基因。利用实时荧光定量PCR技术验证差异基因的时空表达特性；获得拥有自主知识产权的对牡丹响应干旱胁迫有重要功能或调控作用的新基因。最后克隆相关基因进行结构功能的解析，构建载体转化拟南芥进行功能验证。研究结果将在转录组水平上，为牡丹响应干旱胁迫分子机制的研究提供理论依据，并对通过基因工程手段提高牡丹抗旱性，开展牡丹分子育种工作，扩大牡丹种植范围具有重要意义。

[方法] 通过盆栽试验比较两个牡丹品种在干旱及复水诱导下的生理生化、光合性能变化，结合高通量测序技术，从转录水平对比其基因表达差异，挖掘响应干旱胁迫的特异候选基因，利用RT-qPCR验证差异基因表达特性，筛选对干旱胁迫有重要调控作用的基因；开发EST-SSR分子标记，并验证其在不同品种牡丹中的有效性。.[结果] 牡丹品种‘洛阳红’具有耐旱性，而‘乌龙捧盛’属于干旱敏感型牡丹品种。研究挖掘了81, 725个差异表达基因，RT-qPCR验证了20个高度差异表达基因的特性，其中响应干旱胁迫基因12个未知功能基因8个。实验共开发EST-SSR 分子标记7033个，对其中随机挑选额100个分子标记在31个具有不同花色表型的中原牡丹品种中进行了验证，其中有25个EST-SSR分子标记具有多态性。.[结论] 转录水平上挖掘的差异表达基因对通过基因工程手段开展牡丹抗旱分子育种工作具有指导意义，开发并经验证的EST-SSR，可以用作抗旱分子辅助育种的选择标记，同时对庞大的牡丹品种及纯度鉴定具有参考价值。