转录因子PyMYB10与PyMYB114对梨花青苷合成调控研究

Colour is an important agronomic trait of pear and anthocyanin concentration is an important determinant of the colour of red pear.Improving anthocyanin content of fruit has great significance to pear colour and nutritive value. Based on the cloning and characterization of an anthocyanin regulatory gene, PyMYB10, in this research, we will characterize a new gene PyMYB114 which might also be responsible for anthocyanin biosynthesis regulation in pear by RACE-PCR; To analyze the correlation between the expression of PyMYB114 and pear anthocyanin levels,the temporal and spatial regulation patterns of the expression of PyMYB114 and anthocyanin content of pear will also be investigated using Real-time PCR and HPLC respectivly; and function of PyMYB114 in the regulation of the anthocyanin biosynthesis will be further tested by introducing PyMYB114 cDNA under the transcriptional control of the cauli?ower mosaic virus 35S promoter into two heterologous systems: Arabidopsis plants and cultured pear cells. Meanwhile, yeast two-hybrid and dual luciferase assay will be used to find out whether PyMYB10, PyMYB114 could interact with BHLH and with WD40 to regulate the transcripts of anthocyanin structrual genes. The results will enrich the regulation theory of anthocyanin biosynthesis in pear, and also, has great significance to pear colour improvement.

色泽是梨果实重要的农艺性状，花青苷是梨呈现红色色泽的重要成分。在分离梨PyMYB10基因，明确其花青苷调控功能的基础上，本项目拟继续利用RACE-PCR技术对前期获得的可能参与花青苷调控的PyMYB114 cDNA片断进行全长克隆，利用Real-time PCR分析PyMYB114基因在梨中的时空表达模式，利用HPLC技术检测花青苷含量，研究该基因表达水平与花青苷含量的相关关系；利用转基因技术分析其过量表达对拟南芥未成熟种子及梨愈伤组织花青苷合成的影响。同时，利用酵母双杂交及双荧光素酶瞬时表达技术分析PyMYB10、PyMYB114与BHLH及WD40对花青苷合成酶编码基因的协同调控作用。通过以上研究探讨PyMYB114基因的花青苷调控功能以及PyMYB10和PyMYB114调控花青苷合成的分子机制。研究结果不仅能丰富梨花青合成调控理论，而且对梨色泽改良具有非常重要的意义。

色泽是梨果实重要的农艺性状，花青苷是梨呈红色色泽的重要成分。本研究利用RACE-PCR技术克隆得到花青苷调控基因PyMYB10的同源基因PyMYB10.1。序列比对分析发现，PyMYB10.1氨基酸序列与PyMYB10具有较高的一致性。基因表达分析表明，PyMYB10.1和PyMYB10 在富含花青苷的幼叶、花和果皮中大量表达，两者响应光处理的基因表达量趋势与花青苷含量的变化趋势相似。酵母双杂交实验和双分子荧光互补实验表明，PyMYB10.1、 PyMYB10与PybHLH蛋白存在相互作用。烟草双萤光素酶瞬时表达实验表明bHLHs能够增强PyMYB10.1和PyMYB10对花青苷结构基因的调控能力，推测PyMYB10.1、PyMYB10与bHLH形成蛋白复合体调控梨花青苷的合成。研究结果不仅丰富了梨的花青素调控理论，而且对梨色泽改良具有重要的意义。