低温条件下SPLs基因调控牡丹童期的分子机理

The juvenile period plays an important role in tree peony breeding process. The previous results showed that the juvenile period of tree peony could be shortened under low temperature condition and the expression profiles of SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) genes are different in tree peony at different ages. In this project, molecular mechanism of SPL genes regulating the juvenile period of tree peony under low temperature condition will be investigated by using ‘Nongzhishuang’ with shortened juvenile period, and wild species Paeonia ostii and P. delavayi materials. The full length of miR156-targeted SPL genes is cloned first by rapid amplification of cDNA ends (RACE) and allele specific polymerase chain reaction (AS-PCR), and the allelic variation of SPLs from three tree peony species is analyzed. Then the artificial SPLs are constructed. The artificial SPL genes are ligated to pCAMBIA2301G vector to identify their genes function related to regulate plant juvenile period by Arabidopsis genetic transformation. The upstream genes regulating SPLs and the specific binding sites of interested transcription factor genes to the promoters of SPLs are determined by yeast one hybrid (Y1H) and electrophoretic mobility shift assay (EMSA). The relationship between the dynamic transcription expression patterns of SPL genes and their upstream and downstream genes and tree peony juvenile period are constructed by using tree peony at different ages. The low temperature responding mechanism of SPLs is studied by transgenic Arabidopsis and tree peony treated by low temperature. Finally, molecular mechanism of SPL genes regulating juvenile period of tree peony under low temperature condition will be deciphered according to the results obtained in our project. This investigation will provide important theoretical basis for tree peony breeding.

牡丹童期相对较长，严重制约育种进程。前期研究发现低温处理可缩短牡丹童期、SPLs基因在不同年龄阶段的牡丹中表达存在差异。本项目拟利用课题组培育童期较短的牡丹品种‘秾之双’和牡丹野生种‘凤丹’和‘紫牡丹’为实验材料，研究低温条件下SPLs调控牡丹童期的分子机理。首先根据前期已获得SPLs的EST片段，利用RACE技术克隆得到其全长，并探讨其在3个牡丹种中等位变异情况，通过转化拟南芥明确SPLs基因在植物童期调控中可能的生物学功能；采用酵母单杂技术和凝胶阻滞方法，确定SPLs上游调控基因及其与SPLs启动子的特异结合位点；结合已有SPLs的下游基因研究，利用不同年龄阶段的牡丹材料以及不同低温处理的转基因拟南芥和牡丹材料，探讨SPLs及其上、下游基因动态表达特征与牡丹童期的关系以及SPLs基因对低温响应的机理；最后，明确低温条件下SPLs基因调控牡丹童期的分子机理，为牡丹育种提供理论依据。

牡丹童期较长，大大影响牡丹育种进程。前期研究发现低温可缩短牡丹童期，SPLs基因在不同株龄牡丹花芽中存在表达差异。本研究克隆得到16个SPLs基因，基因结构分析表明16个基因均具有保守的SBP结构域，其中9个基因具有miR156的靶位点；基因等位变异分析表明，除了SPL8和SPL10以外，其他SPLs基因都存在丰富的等位变异。不同组织器官和株龄花芽中的表达分析表明，不同基因表达具有组织器官特异性，SPL2、SPL4、SPL8、SPL9和SPL13A2等基因随着牡丹株龄的增加其表达量也逐渐增加，SPL9可能是调控童期的主要基因。利用定点PCR的方法将SPL9基因的miR156靶位点进行了定点突变并构建表达载体进行拟南芥遗传转化，结果表明SPL9可以通过促进下游基因SOC1、LFY、AP1和FT基因的表达缩短拟南芥童期。以紫牡丹为实验材料详细研究了低温条件下SPLs基因调控牡丹童期的分子机理，研究发现一年生株龄的牡丹处在幼年期、三年生株龄牡丹处在成年期，二年生株龄牡丹处在幼年期向成年期的转变期，较大的昼夜温差可以促进牡丹可溶性糖和淀粉的积累，糖和淀粉的大量积累会抑制miR156a的表达，miR156a表达量的减少会解除其对PdSPL9的抑制，PdSPL9的大量表达会促进下游调控基因miR172d和LFY基因的表达，进而实现牡丹幼年期向成年期转变、花芽分化和开花。低温也会促进PdSPL9及开花基因SOC1、AP1、LFY和FT的表达而促进牡丹开花。相关研究可为缩短牡丹童期提供理论依据。