红皮梨花青苷合成调控转录因子PyMYB9和PyMADS18的功能及作用机制

Red pear is currently attractive by customers and possessing extensive development potential in market. As we know,the red-skin color of pear fruit is caused by biosythesis and accumulation of anthocyanins. We founded that transcription factors (TFs) which regulate anthocyanin biosynthesis were important effectors for coloration of pear fruit. In the former research, we obtained the new TFs PyMADS18 and PyMYB9, which effectes anthocyanin biosynthesis significantly,by cDNA-AFLP and whole genome-wide TFs selection. However, the function and regulation mechanism of new genes are still unclear. In this project, the relationship between TF genes mutant and coloration of pear will be study through the comparation of whole length sequence of gene and promoters; the gene function will be identified by using over-expression and RNAi silencing in tobacoo plant and callus of pear;In addition, the proteins which interact with the PyMADS18 and PyMYB9 transcription factor and regulated genes will be screened by using the yeast two-hybrid system and co-immunoprecipitation techniques,then the regulatory network and regulation mechanism of new TF genes will be explored and declared. The results will not only help to elucidate the key factors which regulate the anthocyanin biosynthesis and complete the theory of fruit color development, but also help to promote the process of molecular breeding in red pear and provide necessary gene resources.

红皮梨是目前市场上极具吸引力和发展潜力的果品类型，其果皮红色主要由花青苷的合成与积累所致。项目组前期研究发现花青苷合成的转录调控因子是影响梨果皮着色的重要原因，并利用cDNA-AFLP 以及全基因组范围转录因子筛选获得了调控花青苷合成的两个新基因PyMADS18和PyMYB9，但是其功能及作用机制尚不明确。本项目拟通过对梨色泽突变体（红色/绿色）的PyMADS18和PyMYB9基因全长及启动子序列比较，明确序列结构变化对色泽发育的影响；并通过烟草和梨愈伤组织的RNAi及过量表达转基因鉴定相应的生物功能；同时，利用酵母双杂交及免疫共沉淀等技术分析转录因子互作蛋白和调控基因，从而明确克隆的两个新转录因子调控网络以及对花青苷合成结构基因的调控机制。本研究不仅有利于阐明梨花青苷合成调控的关键因子，完善果皮色泽发育和调控的科学理论，同时也将为推动红皮梨分子遗传改良提供极有价值的指导和必要的基因资源。

红皮梨因鲜艳的外观特征和富含花青苷的保健功能而得到消费者的青睐。但目前我国品质优良的红皮梨较少，且现有的红皮梨常常存在因环境条件影响不着色或者着色不稳定等普遍问题。本项目针对红皮梨着色的分子机制和重要功能基因开展了系统研究，主要结果如下： 利用‘八月红’与‘砀山酥梨’杂交F1代群体，通过遗传图谱构建和QTL定位，将色泽性状定位于LG5上，并对应染色体区域筛选到控制红色性状重要候选基因PyMYB114(原命名PyMYB9)。进化树构建结果显示，该基因与拟南芥中控制色泽的MYB基因家族成员聚在一组。结合RNA-seq数据筛选到与PyMYB114共表达的基因PyERF3和PybHLH3。双荧光素酶报告系统和酵母双杂交试验证明了PyMYB114分别与 PyERF3和PybHLH3互作，并激活花青苷合成途径的结构基因PyDFR、PyANS和PyUFGT的启动子，增强花青苷的生物合成；且超表达PyMYB114和互做因子瞬时转化促进了草莓和梨果实的花青苷合成及果实着色。在此基础上，提出了PyMYB114与PyERF3和PybHLH3形成调控复合体调控梨果实着色的工作模型。同时，研究发现PyMADS11（原命名PyMADS18）和PyMADS12与梨果皮花青苷含量呈正相关，瞬时转化梨果实PyMADS11和PyMADS12促进了花青苷合成相关转录因子和结构基因的表达，从而促进了果实着色。