牡丹miR172d和PsAP2调控花芽内休眠进程的分子机制

MicroRNAs are important factor to regulate gene expression, and the interactions between miRNAs and their targets play core roles in regulation of many physiological process. Antiseason forcing culture is one of the most important contents of tree peony (Paeonia suffruticosa Andr.) industry in china, and the core technology is to accelerate endo-dormancy release of flower bud and flowering early after sufficient chilling treatments. Therefore, the analysis of dormancy mechanism is the base to resolve the practice problems of tree peony industry. One EST sequence of AP2 gene using 454 sequencing and microarray was obtained, and the protein of AP2 was also screened using dimensional electrophoresis and mass spectrum, which were differential expressed and their expression trends during endo-dormancy release were consistent. At the same time one miR172d was obtained after small RNA sequencing and microarray. The full cDNA of PsAP2 had been obtained using RACE amplification method. MiR172d was speculated to be complementary with the exon3 domain of PsAP2 sequence near the stop codon by RNAhybrid software. The expression patterns of PsAP2 and miR172d during dormancy release were corresponding. These results suggested that PsAP2 had potential target relationship with miR172d. This project aims to analyze the target relationship between miR172d and PsAP2 during dormancy release, and make it certein that if miR172d directly acts on PsAP2 and how miR172d regulate PsAP2 in order to affect the dormancy release.The expression patterns of PsAP2,miR172d and the downstream genes, PsFT and PsSOC1, will be detected during dormancy release. The method about miR172s regulating its target gene PsAP2 will be detected using western blot and Northern blot. The time changes of dormancy release will be observed and the expression patterns of PsFT and PsSOC1 will be analyzed using qRT-PCR and Northern blot methods after VIGS technology mediated PsAP2-silencing. At the same time, the over-expression vectors of miR172d and PsAP2 will be constructed and transfect wild Arabidopsis, and the vernalization time and the expression patterns of two downstream genes (AtFT and AtSOC1) will be obtained in over-expression Arabidopsis.In addition, the promoter of the PsFT will be cloned using TAIL-PCR, and the intraction between PsAP2 and PsFT will be analyzed using yeast hybrid technology and electrophoretic mobility shift assay (EMSA). These results will offer theory base of miRNAs regulated the mechanism of dormancy release in tree peony, and provide candidate genes and theoretical direction for forcing culture and molecular breeding.

春节催花是牡丹产业链条的重要内容，其技术核心是低温解除花芽内休眠，因此深入解析内休眠机理是解决催花不良等生产问题的前提。MicroRNA是重要的基因表达调控因子，其与靶基因互作在植物多个生理过程中具有核心调控作用。课题组利用小RNA测序和转录组分析筛选到休眠进程相关的1个miR172d和AP2基因EST；RACE扩增了PsAP2全长cDNA。生物软件分析和RT-PCR结果暗示两者存在潜在的靶标关系。本项目拟以此为切入点，鉴定miR172d和PsAP2的靶标关系以及miR172d对PsAP2的调控方式；进一步分析VIGS沉默PsAP2后牡丹花芽休眠进程和下游基因PsFT、PsSOC1的表达变化，结合超表达miR172d、PsAP2拟南芥的春化时间和AtFT、AtSOC1基因表达分析，解析miR172d和PsAP2调控内休眠的机理。这将为进一步阐明miRNAs在内休眠进程中的功能奠定理论基础。

牡丹(Paeonia suffruticosa Andr.)是原产于我国的特有名贵花卉，具有很高的观赏价值、药用价值和经济价值。春节催花是牡丹产业的主要内容之一。牡丹反季节催花的关键问题是解除花芽内休眠，促使牡丹成功提前开花。MicroRNA是重要的基因表达调控因子，其与靶基因互作在植物多个生理过程中具有重要调控作用。课题组前期利用小RNA测序筛选到112个差异表达的小RNA，其中包含miR172家族成员；RACE扩增了PsAP2全长cDNA，实时定量结果表明其在牡丹休眠解除中呈上调趋势。通过生物软件分析，暗示PsmiR172e和PsAP2两者存在潜在的靶标关系。本课题内容以此为切入点，克隆了PsFT基因及启动子序列，酵母单杂交实验表明PsAP2蛋白能与PsFT启动子结合；转PsAP2基因拟南芥表型及分子鉴定表明，PsAP2抑制开花，且负调控FT基因表达。实时定量和原位杂交分析PsmiR172s家族成员的表达模式，结果表明在低温解除花芽休眠过程中，PsmiR172s基因不同成员的表达量不同，其中PsmiR172e在低温7 d达到最大值，之后呈下降趋势，与PsAP2基因的表达趋势相反。通过RT-PCR和Western blot实验，预测PsmiR172e和PsAP2存在靶标关系。利用置换法构建牡丹PsmiR172e与GUS融合表达载体，构建了PsAP2和mPsAP2（靶位点同义碱基突变）与GUS融合表达载体，分别与PsmiR172e::GUS载体共转化烟草叶片。结果表明PsAP2与PsmiR172e共转化烟草后的GUS活性显著降低，表明PsmiR172e和PsAP2存在靶标关系。GUS活性的测定结果与GUS的组织化学染色结果相一致。5' RLM RACE实验进一步分析PsmiR172e在PsAP2中的剪切位点，在18个阳性克隆中有10个在距PsmiR172e 5’端的第9（G）和第10个碱（A）之间的位置被剪切，与其他物种中的检测结果一致。通过转基因拟南芥表型分析（开花时间和花期等）进一步验证了PsmiR172e与PsAP2的靶标关系。过表达PsmiR172e拟南芥开花时间提前2 d，花期缩短1 d。结合下游开花基因FT的表达情况，表明PsmiR172e基因促进开花，具有功能保守性。研究结果将为进一步阐明miRNAs在内休眠进程中的功能奠定理论基础。