山茶miR156及其靶基因SPLs调控重瓣花发育的分子机制

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.

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。

本项目完成了山茶及其重瓣品种中microRNA（miRNA）的鉴定和表达研究，结合小RNA深度测序和降解组测序，对花器官组织特异性表达的miRNA及其靶基因开展了克隆、表达及其功能鉴定研究。利用重瓣山茶内轮花器官的形态变异，针对的miR156-SPLs组合，对MIR156家族成员及其下游基因SPL类转录因子（SQUAMOSA Promoter Binding Like）开展了验证；同时利用山茶中愈伤表达体系和花瓣中的瞬时表达体系，研究了miR156-SPLs调控花器官组织发育的分子机制，揭示出miR156介导的基因转录调控是重瓣山茶内轮花器官精细化发育的调控节点。同时，利用MIRNA基因及其靶基因位点的系统性发掘，发现miR5179和miR172在花器官发育调控中具有重要的调控作用。项目总体完成了既定的研究目标，主要包括以下几个方面：1）完成山茶全基因组内miRNA的鉴定、家族分析、进化关系等研究；2）分析了单瓣、重瓣山茶花器官中miRNA表达模式分析，筛选出与重瓣花器官发育相关的miRNA基因；3）利用降解组测序和靶基因表达相关分析，完成了miRNA基因的靶位点基因的鉴定与功能分析，利用LNA探针原位杂交实验研究了miR156、miR5179、miR172的成熟miRNA在花发育不同阶段的组织特异性表达模式；4）针对内轮花瓣、内轮雄蕊的差异表达筛选，重点针对miR156/SPLs和miR5179/DEF的调控组合进行了异源表达、山茶瞬时表达、靶位点验证等研究，解析了miR156-SPLs调控节点对花型发育的分子机制。本研究以重瓣山茶花的内轮花器官的形成为研究对象，揭示出特异性表达的miRNA基因的功能，丰富了花型建成的理论，为进一步阐明不同重瓣花的发育调控机制奠定基础。本项目成果在《Journal of Experimental Botany》等杂志上发表研究论文7篇、申请国家发明专利1项，支持项目培养了2名硕士研究生。