梨miR156及其靶基因SPLs介导花青苷合成光信号应答的分子机制

As we known, miR156 and its target gene SPLs are involved in regulation of Juvenile phase transformation in fruit, the function of mediating anthocyanin biosynthesis in fruit still do not reported. Light is one of the most important environmental factor which regulate the anthocyanin biosynthesis, miR156 and its target gene SPLs were interfere anthocyanin biosynthesis responsing to light signal in previous study of our project group. Typical red pears including Oriental pear and Occidental pear with different responded patterns to light signal will be used in this study, separate the candidate gene of SPLs and analyze the functions of these genes combining the bioinformatics analysis and subcellular localization. At the same time, test the corresponding relationship between miR156 and its target gene SPLs with the method of degradome sequencing and 5’RACE. After that, it will be further identified interaction relationship between SPLs and MYB-bHLH-WD40 protein complex using Y2H and CO-IP methods in vivo and in vitro. In the end analyze the molecular mechanism response to light signal of miR156 and its target gene SPLs mediate anthocyanin biosynthesis, employing transfer genes in Arabidopsis and transient expression in fruit. The research will be helpful to clarify the molecular mechanism response to light signal of miR156 mediate anthocyanin biosynthesis, enrich the theory of molecular mechanism of fruit anthocyanin biosynthesis，and give us good guidance for producing red pear fruit with attractive appearance.

miR156参与果树童期转变调控被大家所熟知，但其介导果实花青苷合成的功能尚见未报道。光照是调控果实花青苷的合成最重要的环境因子之一，项目组前期研究发现miR156及其靶基因SPLs介入花青苷合成的光信号应答，本项目拟以具有不同光应答效应红色砂梨和西洋梨品种为试材，分离SPL家族关键成员，结合生物信息学分析和亚细胞定位，对其功能作出预测；然后利用降解组测序和5’RACE的方法验证miR156与其靶基因SPLs的对应关系；依照前期提出的工作模式图，利用Y2H和CO-IP技术验证SPLs与MYB-bHLH-WD40蛋白复合体在体内外的互作调控关系；再利用拟南芥转基因和梨果实瞬时表达验证两者的功能。本申请项目的实施有助于阐明miR156介导花青苷合成光信号应答的分子机制，丰富光诱导果实花青苷合成的生物学理论，对梨果皮色泽的遗传改良，指导优质红梨生产中的着色调控，均具有重要理论和实践意义。

miR156是植物上广泛存在并进化上非常保守的miRNA,并且通过作用其靶基因SPL来调控植物生长发育的各种过程。本项目，通过sRNA和降解组测序，发现在梨果皮中表达的27个miR156家族成员，并发现4个可以被miR156降解的SPL基因。通过梨全基因组SPL家族成员鉴定，发现19个PpSPL成员，其中11个成员拥有miR156的剪切位点。. 果皮着色过程中，miR156表达上调，而PpSPL基因中，PpSPL2、PpSPL5、PpSPL7、PpSPL9、PpSPL10、PpSPL13、PpSPL16、PpSPL17和PpSPL18 的表达下调。酵母双杂结果显示，梨上调控花青苷合成的3类转录因子可以形成MYB/bHLH/WD40转录因子复合体，并且PpSPL10和PpSPL13与PyMYB10存在互作。结合前人的研究和本项目的研究结果，提出了以下假说：黑暗中，由于miR156的低表达，高表达的SPL通过破坏MYB/bHLH/WD40蛋白复合体的形成，进而抑制CHS、ANS和 UFGT的表达来负调控花青苷的合成。去袋进行重曝光后，miR156上调表达并降解SPL基因，使的MYB/bHLH/WD40蛋白复合体重新形成，从而促进了花青苷合成相关结构基因的表达，并最终诱导花青苷的合成。