芍药种子下胚轴休眠与萌发相关调控基因的克隆及功能分析

Both epicotyl and hypocotyl of Paeonia lactiflora seeds possess the characteristic of double dormancy. The time duration from sowing to emerging for Paeonia lactiflora seeds is approximately 6-7 months under the natural condition and the vegetative cycle is long. Because the dormancy of seeds is not broken or not broken thoroughly in the process of seed propagation, germination rate is decreased and which affects cultivation and production, especially affects the process of cross-breeding. Hypocotyl dormancy is the first step to inhibit seed germination. According to the restriction fragments detected on denaturing polyacrylamide gel during our project preparatory, we found that there were larger differences between the genes of hypocotyl before and after germination than the genes of epicotyl before and after germination. On this basis, our project intends to use transcriptome sequencing technology of De novo to get the gene expression profiles of Paeonia lactiflora seeds, analyze gene expression differences of hypocotyl of Paeonia lactiflora seeds before and after germination on base of Tag-seq data and use real-time quantitative RT-PCR technology to validate and analyze differently expressed genes. Based on the above studies, we use Arabidopsis as material to analyze the function of up-regulation and down-regulation genes by the over expression of genes during germination of Paeonia lactiflora seeds and explore the mechanisms that lead to gene differential expression. The project aims at revealing the molecular mechanisms of seed dormancy and germination of Paeonia lactiflora, which plays an important role on improving the efficiency of cultivation and production, accelerating the process of cultivating new varieties and strengthening protection and utilization of wild resources.

芍药种子具有上、下胚轴双重休眠的特性，自然条件下从播种到出苗需要6-7个月的时间，生长周期长。种子繁殖过程中常因休眠未解除或解除不彻底而使萌芽率降低，影响栽培生产，尤其影响杂交育种工作的进程。下胚轴休眠是抑制芍药种子萌发的第一步，项目前期通过变性聚丙烯酰胺凝胶电泳检测发现，打破下胚轴休眠前后的差异要远远高于上胚轴。在此基础上，本项目拟利用De novo 转录组测序技术获得芍药种子下胚轴萌发前后的基因表达谱，基于Tag-seq 数据分析芍药种子下胚轴萌发前后基因表达差异，并利用实时定量RT-PCR 技术对差异表达的基因进行验证分析。在上述研究基础上，以拟南芥为试材通过基因过量表达，分析种子萌发过程中上调和下调表达基因的功能，探讨导致其差异表达的机制。本项目旨在揭示芍药种子休眠与萌发的分子机理，对于提高栽培生产效率、加快新品种培育进程及加强野生资源保护与利用等方面具有重要意义。

芍药种子具有上、下胚轴双重休眠的特性，自然条件下从播种到出苗需要6-7个月的时间，生长周期长。种子繁殖过程中常因休眠未解除或解除不彻底而使萌芽率降低，影响栽培生产，尤其影响杂交育种工作的进程。下胚轴休眠解除是打破芍药种子整个休眠的起始和关键过程，本项目完成了芍药种子下胚轴萌发的转录组测序，共得到1795个差异基因，并应用qRT-PCR对转录组数据进行了验证，表明测序数据准确。在此基础上，筛选出与下胚轴萌发相关的关键基因，对功能进行初步研究：同源克隆芍药种子下胚轴休眠解除关键基因PlGAI1，并进行原核表达，克隆了PlGAI1的启动子并检测其具有转录活性，同时对芍药休眠萌发相关基因PlPP2C进行了研究；此外，初步建立了以芍药胚性愈伤组织为遗传转化受体材料的农杆菌侵染体系。芍药的分子基础研究十分薄弱，对其遗传背景了解很少，本项目在一定程度上揭示了芍药种子下胚轴休眠解除分子机理，并对部分关键基因功能进行分析，为完善芍药的分子信息及挖掘潜在的新基因提供了丰富的数据，未来课题组将在已有研究基础上开展更为深入的研究。