毛果杨miR1444a在酚类化合物及木质素产生过程中的作用研究

Phenolic compounds play significant roles in plant response to biotic and abiotic stresses. Lignin, a phenolic derivative, is the main barrier in paper pulping and bioethanol production. Based on our previous results, it is very likely that miR1444a regulates the production of phenolic compounds and lignin in Populus trichocarpa through targeting polyphenol oxidase genes for cleavage. With the aim to test this hypothesis, we propose to use P. trhichocarpa, which has the whole genome sequence available, as experimental materials. We will construct miR1444a overexpression vector and then introduce it into P. trichocarpa. The content and composition of phenolic compounds and lignin will be comparatively analyzed in transgenics and non-transgenics. High-throughput degradome sequencing and RNA-seq will be performed for transcriptome-wide analysis of genes directly cleaved or indirectly regulated by miR1444a. All of the data obtained will be further comprehensively analyzed for elucidating the relationships among miR1444a expression level, protein-coding gene expression level, the content and composition of phenolic compounds, and the content and composition of lignin in P. trichocarpa. We expect that the regulatory network of miR1444a will be constructed at the whole genome level and the regulatory mechanism of miR1444a on the production of phenolic compounds and lignin will be finally elucidated. The results will provide useful information for elucidating the genetic regulatory mechanism of phenolic compounds and lignin production and will serve as a basis for controlling phenolic compounds and lignin production through genetic engineering approaches.

酚类化合物在植物抗病、抗逆过程中发挥着重要作用，酚类衍生物木质素还是造纸和利用生物质生产酒精的主要屏障。我们前期的研究表明，miR1444a很可能介导多酚氧化酶参与调控毛果杨酚类化合物和木质素的产生。为了验证这一假设，本项目将以基因组全序列测定已完成的毛果杨为试材，构建miR1444a过表达载体，遗传转化，获得miR1444a过表达的转基因毛果杨；比较分析转基因和非转基因植物体中酚类化合物和木质素的组分和含量变化，结合转录组和降解组的高通量分析，找出miR1444a表达量、基因表达量、酚类化合物的组成与含量、木质素的组成与含量之间的关系；从全基因组水平构建miR1444a调控的基因网络，验证miR1444a介导多酚氧化酶调控酚类化合物和木质素产生的假设，阐明其分子机制，为彻底揭示毛果杨酚类化合物和木质素产生的遗传调控机制和利用基因工程手段调控酚类化合物和木质素的产生奠定基础。

酚类化合物往往是植物体内重要的次生代谢物。它们不仅在植物抗病、抗逆过程中发挥着作用，也与人类的生产生活密切相关。研究酚类化合物生物合成的调控机理，具有重要的意义。植物微小RNA是一类长度约为21个碱基、在植物体内发挥重要调控作用的非蛋白质编码RNA。本项目（1）克隆了毛果杨ptr-MIR1444a基因，构建了过表达载体，通过遗传转化，获得了转基因株系并进行了详细的分子鉴定和酚类化合物的分析，证实ptr-miR1444a可介导PPO基因调控酚类化合物的生物合成；（2）系统鉴定多种植物的PPO基因家族和靶向PPO的miRNA，发现miRNA对PPO的调控具有物种的特异性，毛果杨中含有ptr-MIR1444a和ptr-MIR1444b等2条ptr-MIR1444基因，二者既有相同的靶基因又有不同的靶基因。在此基础上，开展了ptr-MIR1444b基因的克隆、过表达载体构建与遗传转化研究，研究结果为阐明ptr-miR1444b的功能及作用机制奠定基础，有助于解析PPO靶基因家族不同成员在酚类化合物合成中的作用；（3）系统地比较分析了在杨柳科、唇形目和葡萄属植物中靶向PPO基因的miR1444、miR12112和miR058及其靶基因，对靶向PPO基因的miRNA的起源和进化过程进行了详细分析，提出了MIRNA基因起源和进化的新模式；（4）共获得或鉴定蛋白质编码基因序列58条、MIRNA基因或前体序列115条、双元表达载体3个、转基因株系9个，在SCI期刊发表论文6篇，SCI影响因子总和27.241，参编英文专著1部，有4名博士后和博士生参与了研究工作，参加国内国际学术会议2人次，取得了较好的结果。