丹参特异miR12112在酚类化合物生物合成中的调控作用研究

Salvia miltiorrhiza Bunge, known as danshen, is a well-known material of traditional Chinese medicine (TCM) with whole genome sequence available. It is emerging as a model system for TCM studies. Phenolic compounds are a group of bioactive ingredients. The regulatory mechanism of phenolic compound biosynthesis in S. miltiorrhiza is largely unknown. miR12112 is a danshen-specific, function-unknown miRNA first reported by our research group. Based on our previous results, it is very likely that miR12112 regulates the production of phenolic compounds in S. miltiorrhiza through targeting polyphenol oxidase SmPPO genes for cleavage. With the aim to test this hypothesis, we propose to clone the full-length MIR12112 and its targets SmPPO1, SmPPO10, SmPPO12 and SmPPO16. Vectors over-expressing miR12112, SmPPO1, SmPPO10, SmPPO12 and SmPPO16 will be constructed and introduce into S. miltiorrhiza. Transgenic plants with miR12112 over-expression and SmPPO down-regulation and those with SmPPO over-expression will be comparatively analyzed with non-transgenic wildtype plants for transcriptomes and phenolic compounds. All data obtained will be comprehensively analyzed for the relationships among the expression levels of miR12112, SmPPOs and genes indirectly regulated by miR12112, and the content and composition of phenolic compounds. We expect that the regulatory network of miR12112 will be finally constructed at the whole genome level. The results will serve as a basis for development of new S. miltiorrhiza lines with high content of bioactive phenolic compounds.

丹参是全基因组序列测定已完成的传统中药材，正成为模式药用植物。酚类化合物是丹参中一类重要的药用活性成分，其生物合成的调控机制还很不清楚。miR12112为我们课题组首先报道发现的、功能有待研究的丹参特异miRNA。前期的研究表明，它很可能介导丹参多酚氧化酶SmPPO基因调控酚类化合物的产生。为验证这一假设，本项目将克隆MIR12112及其靶基因SmPPO1、SmPPO10、SmPPO12和SmPPO16，构建过表达载体，转化丹参，比较分析miR12112过表达引起SmPPO基因下调的转基因丹参、SmPPO过表达的转基因丹参以及非转基因丹参体内特别是根中的转录组和酚类化合物，找出miR12112表达量、SmPPO表达量、受miR12112间接影响的基因表达量、酚类化合物的组成与含量之间的关系，从全基因组水平构建miR12112的遗传调控网络，为培育酚类化合物含量较高的丹参新品系奠定基础。

酚类化合物是植物体内一类重要的活性成分，揭示其生物合成途径和调控机制具有重要的理论和实际意义。酚类化合物生物合成过程中，有大量的关键酶参与。植物微小RNA通过靶向切割这些关键酶基因的转录本调控酚类化合物的合成。本项目（1）从组学水平系统鉴定了79条植物MIR12112基因，发现MIR12112很可能只存在于唇形目植物中，是唇形目特异的MIRNA基因，进而探讨了MIR12112基因起源和进化的分子机制；（2）克隆了丹参Smi-MIR12112a基因及其靶基因SmPPO，构建了基因过表达或敲除载体，开展了丹参的遗传转化及转基因植物的分析，证实smi-MIR12112a介导SmPPO基因参与了酚类化合物生物合成的调控；（3）发现受miR397调控的漆酶（SmLAC）基因与受miR12112调控的SmPPO基因一样，参与了丹参酚类化合物的生物合成。其中，SmLAC1既能催化B型原花青素的聚合，也能催化A型原花青素的聚合。研究结果解决了几十年来悬而未决的有关原花青素聚合机制的科学问题，也使原花青素合成生物学研究成为可能；（4）完善、整理和系统总结了一些功能相关基因和基因家族的鉴定与分析工作，为彻底阐明酚类化合物的生物合成及调控机制奠定了良好基础；（5）发表论文SCI论文12篇，最新影响因子总和50.341，主编中文专著1部、英文专著1部，参加国际学术会议并作报告2人次；（6）博士后出站2名，博士生毕业4名、在读5名。