LsMYBs转录因子调控换锦花红蓝复色花形成的分子机制

Anthocyanin is an important plant pigment and the biosynthesis of anthocyanin can be regulated by R2R3-MYBs transcription factors. However, the formation mechanism of multiple colored flowers is complex. Therefore, it is of great significance to explore the formation mechanism of multiple colored flowers. L. sprengeri (Lycoris genus) has unique flower pattern with red and blue color on petals. But the formation mechanism of multiple color flowers is yet to know. In this study, we will use L. sprengeri multiple color flowers as our materials. Preliminary data indicated that the transcription factors LsMYBs ( LsMYB1, LsMYB4 and LsMYB5 ) possibly affected the flower color phenotypes in our previous research. The molecular regulatory mechanism of multiple color flowers will be further studied by our group using molecular biology, function genomics, and physiological and biochemical research methods like qRT-PCR, RNA fluorescence in situ hybridization, yeast one hybrid, L.sprengeri petal transient expression technique, electrophoretic mobility shift assay (EMSA), DNase I footprint and allogenic transformation in petunia. The results are expected to reveal the plant multiple color flowers formation and regulatory network and molecular targets DNA elements for genetic improvement of flower color through genes engineering methods, which will provide a theoretical basis for artificial creation and molecular breeding of multiple color flowers in Lycoris species and ornamental plants.

花色素苷是决定植物花色的重要成分，R2R3-MYBs类转录因子可调控花色素苷生物合成，复色花形成的作用机理复杂，探讨复色花形成调控机制具有重要的意义。石蒜属植物换锦花具有独特的红蓝复色花表型。本项目选用课题组拥有的各种红蓝复色花变异表型的换锦花种质为试材，以前期研究发现的可能影响换锦花花色表型的LsMYBs（LsMYB1、LsMYB4和LsMYB5）转录因子为切入点，采用qRT-PCR、RNA荧光原位杂交技术、酵母单杂交技术、换锦花花瓣瞬时表达技术、凝胶阻滞实验和DNase I足迹体外试验以及异源瞬时转化矮牵牛花瓣等分子生物学、功能基因组学及生理生化等研究手段，聚焦于破解LsMYBs对红蓝复色花形成调控的分子机制，有望获得有关植物复色花形成及调控网络的新知识以及可用于花色基因工程遗传改良的分子靶标等DNA元件，为石蒜属植物及更多观赏植物复色花的人工创制和分子育种提供理论基础。

本项目采用细胞学和分子生物学等方法研究LsMYBs对换锦花花色形成调控机理，结果可为石蒜属植物花色育种提供参考。结果如下：.1.花色苷是换锦花呈色主要成分，LsMYB4和 LsMYB5为转录抑制因子；.2.建立LsMYBs的VIGS基因沉默体系，瞬时转化换锦花花瓣发现LsMYB5可能调控LsANS、LsDFR3和LsUFGT，而LsMYB4可能调控LsCHS、LsCHI、LsF3H、LsF3'H、LsANS、LsDFR3和LsUFGT；沉默后换锦花花色苷总量与各基因表达一致；.3.通过Pulldown assay和质谱相结合从换锦花花瓣蛋白粗提液中筛选到与LsMYBs原核蛋白互作的可能有MYB、bHLH、WD repeat、ABC等；.4.LsMYB4在烟草中过表达后，花瓣颜色变浅，转基因株系中花色苷形成相关基因的表达下降，烟草花瓣花色苷含量和基因的表达一致。推测LsMYB4调控靶基因可能为DFR、ANS、UFGT、F3H；获得4个LsDFRs、1个LsANS和1个LsUFGT1和启动子序列；.5.获得到4个LsbHLHs，属于核转录因子，可能参与调控花色苷合成基因表达。.6.酵母单杂交表明LsMYB4能激活LsANS和LsDFR3启动子活性，LsMYB5激活LsF3’H、LsANS和LsDFR3启动子活性。.7.双荧光素酶报告检测LsMYB4能够激活LsANS启动子活性，不激活LsF3’H、LsUFGT1和LsUFGT2启动子活性；LsMYB5不激活4个基因启动子活性。LsMYB4和LsbHLH2复合体可激活LsF3’H和LsANS启动子活性；证明LsMYB4或LsMYB4/LsbHLH2复合体对LsF3’H和LsANS起着调控作用。.8.绘制LsMYB4和LsMYB5调控换锦花复色花色形成的模式图。