目前，一些调控离体苗再生基因已被发掘，其分子机理仍不清楚。我们发现miR159及靶基因MYB65控制拟南芥离体苗再生，其如何控制值得研究。本研究拟利用Col-0、35S::miR159、miR159ab、mMYB65（抗miR159剪切突变体）及myb65开展以下研究：分析上述材料表型及miR159及MYB65时空表达模式，明确其调控的再生时期及其表达定位关系；比较上述材料中WUS和CLV3时空表达模式差异，确定miR159及MYB65何时参与干细胞中心重建。利用RNA-seq和ChIP-seq，筛选候选靶基因。利用EMSA、ChIP、点突变靶基因启动子及植物杂交，验证MYB65与靶基因转录调控关系及遗传作用关系。利用酵母双杂技术发掘MYB65的互作蛋白，利用BiFC、GST-pull down、Co-IP等进一步验证，并确定其遗传学关系，揭示miR159调控离体干细胞中心重建的分子机制。

So far, a set of genes regulating shoot regeneration have been found. However, the molecular mechanism is not clear yet. Our previous study showed that miR159 and MYB65 affected shoot regeneration, the mechanism of which was worth investigating. In this program, we will use Col-0, 35S::miR159, miR159ab, mMYB65 (miR159-resistant version of MYB65) and myb65 mutants to carry out the research as follows: Analyze the phenotype of the above plant lines and spatial-temporal expression patterns of miR159 and MYB65. Conform the stage that miR159 and MYB65 control during shoot regeneration and the regulation and localization relationship of them. Verify the exact time when miR159 and MYB65 control reconstitution of the shoot stem cell niche via spatial and temporal expression profile of WUS and CLV3 of the above plant lines. Screen the candidate target genes via RNA-seq and ChIP-seq. Validate the direct binding of MYB65 to target genes and confirm the genetic relationships of them via EMSA, ChIP, point mutant of promoters and plant crossing. Exploring and confirm the interacted proteins of MYB65 via yeast two hybrid, BiFC, GST-pull down and Co-IP. Validate the genetic relationships of them. The crucial aim of the project is to elucidate how miR159 regulates reconstitution of the shoot stem cell niche from in vitro cultures of Arabidopsis thaliana.