重瓣是栽培山茶花重要的观赏性状。重瓣山茶的内轮器官在属性和形态上存在显著的特殊性，但对其发育调控的分子基础尚无定论。项目组在前期转录组和小RNA的研究中发现，miR156及其靶基因SPLs在重瓣山茶的内轮花器官发育中存在表达差异。为阐明miR156-SPLs在重瓣山茶发育中的调控机理，本申请开展miR156及SPLs靶基因的功能研究。通过基因克隆、靶标位点验证及mRNA原位杂交研究，甄别参与调控内轮花器官发育的miR156-SPLs组合；建立山茶愈伤转化体系，开展异位表达SPLs、靶位点突变的SPLs基因、共同表达miR156前体和SPL靶基因等研究，解析miR156-SPLs涉及的调控网络；进而结合转录组及ChIP-Seq，鉴定下游基因及其转录调控的结合位点。通过研究，揭示miR156-SPLs调控内轮花器官发育的分子途径，阐明花型建成的调控网络，为分子设计定向选育提供理论基础。

Double flower formation is an economically important ornamental trait in cultivated camellias. The inner floral organs of doubled Camellia cultivars display remarkably variations on shape and identity. However, it remains unclear how these double flowers are regulated at the molecular level. Our previous work has demonstrated that the development of double flowers in Camellia may require the coordination of multiple developmental pathways. And the miR156 and its targeted SQUAMOSA Promoter-Binding Protein Like Genes (SPLs) were differentially expressed in the inner floral organs. In order to investigate the functions of ‘miR156-SPLs’ during double flower development, this proposal integrates the studies of transcriptome and small RNA sequencing to understand the functions of miR156-SPLs. Through gene cloning and target sites evaluation, we identify the combinations of miR156 and SPLs which are related to floral development; further, we characterize the functions of miR156 and SPLs by the transgenic Arabidopsis approach; moreover, taking advantage of a callus transformation system in Camellia, we study the miR156 and SPLs by ectopic expression assays; finally we perform transcriptome and chromatin immunoprecipitation sequencing (ChIP-Seq) by using the transformed callus to identify the downstream genes. Taken these results together, we discuss the molecular functions of miR156 and SPLs relevant to the inner floral organ development in doubled Camellia cultivars.