基于转录组学的芍药种子休眠与萌发分子机理研究

Paeonia lactiflora seeds possess the characteristic of double dormancy. The germination characteristic shows the hypocotyl elongation and the radicle emergence when the temperature decrease in autumn, and the dormancy of the epicotyl is broken after low temperature in winter, then the germ comes out of the earth and germinates after temperature rising in spring. Division with long production cycles and low coefficient of propagation is the main mode of propagation of Paeonia lactiflora. Seed reproduction, as the only means to get seedlings of Paeonia lactiflora, is the foundation of asexual reproduction. 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 so long that it brings great difficulties to the artificial cultivation production and the inspection of Paeonia lactiflora seeds. Considering the molecular mechanism of double dormancy of Paeonia lactiflora seeds is unknown so far, our project intends to use transcriptome sequencing technology of De novo to get the gene expression profiles of Paeonia lactiflora embryo, analyze gene expression differences of epicotyl and hypocotyl of Paeonia lactiflora embryo 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 analyze the function of up-regulation and down-regulation genes by the over expression and the silence 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, then establishing a theoretical foundation for shortening the breeding cycles, improving breeding efficiency and solving the problem of the production and forcing culture of Paeonia lactiflora.

芍药种子具有双重休眠的特性，表现为秋季降温时下胚轴伸长，长出胚根，经过冬季低温后上胚轴休眠被破除，春季升温后胚芽出土萌发。芍药繁殖方式以分株为主，但生产周期长，繁殖系数低。种子繁殖是获得芍药实生苗的唯一手段，是无性繁殖的基础。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。对芍药的人工栽培生产和种子检验带来很大的困难。芍药种子的双重休眠分子机理至今未明。本项目拟利用De novo 转录组测序技术获得芍药种胚的基因表达谱，基于Tag-seq 数据分析芍药种胚上、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，通过基因过量表达和沉默来分析芍药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，为缩短芍药牡丹育种周期、提高育种效率及解决芍药生产和促成栽培难题奠定理论基础。

芍药种子具有双重休眠的特性，对芍药的人工栽培生产和种子检验带来很大的困难。本项目利用常规石蜡切片技术找出打破上、下胚轴休眠的关键时期；建立芍药种子cDNA-AFLP分析体系和反应程序；对打破上、下胚轴休眠前后的芍药种子进行cDNA-AFLP分析。为进一步分离、定位、克隆芍药种子休眠基因，揭示芍药种子休眠与萌发的分子机理奠定基础。主要研究内容及结果如下：.（1）利用常规石蜡切片技术观察芍药种子沙藏催芽过程中胚的结构和形态变化，发现15℃沙藏层积45d，芍药种子裂口露白，下胚轴细胞分裂旺盛，是打破下胚轴休眠的关键时期。15℃沙藏根长达到3-4cm后进行4℃低温催芽28d，上胚轴明显伸长，打破了上胚轴休眠。.（2）建立了芍药种子cDNA-AFLP分析优化体系和反应程序。RNAprep pure Plant Kit（天根公司）提取RNA，利用M-MLV RTase cDNA Synthesis Kit（TaKaRa）对总RNA进行双链cDNA的合成。限制性内切酶EcoRⅠ和MseⅠ对cDNA进行37℃ 4h，65℃ 20min酶切后连接EcoRI和MseI接头。连接产物稀释10倍用于预扩增，预扩增的反应程序为94℃ 3min；94℃30s，50℃30s，72℃1min，30个循环；72℃5min。预扩产物稀释10倍作为选择性扩增的模板，优化得到的选扩反应体系为：20μl的反应体系中含5μl模板，10×PCR buffer 2μl，EcoRⅠ特异性引物1μl，MseⅠ特异性引物1μl，dNTP（10mM）0.4μl，Taq DNA Polymerase（2.5U）0.4μl。选择扩增程序：94℃3min；94℃30s，65℃30s（每个循环降0.7℃），72℃1min，13个循环；94℃30s，56℃30s，72℃1min，24个循环；72℃7min。.（3）对打破上、下胚轴休眠前后的芍药种子进行cDNA-AFLP分析，发现打破下胚轴休眠前后共3700条差异条带，打破上胚轴休眠前后共1643条差异条带。

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