VvmiR156及其靶基因VvSBP8在水分亏缺诱导葡萄果实花色苷积累中的作用机理

Anthocyanins are a kind of important color compounds in grape berry and wine. The moderate water deficit can induce the their accumulation in grape berry, but the mechanism is not clear. MicroR156 is a small conserved non-coding RNAs that coordinates the relationship between development and abiotic stress tolerance in plants. In our previous study, it has been found that VvmiR156 and its targeted VvSBP8 expression level is linear with the anthocyanins content in grape berry during ripening when vines were treated with water deficit. Herein, we will overexpress VvSBP8 to red flowering tobacco species Nicotiana Xsanderae and transiently transfer its interference vectors to grape berry skins using virus induced gene silencing to confirm the function of VvSBP8 negatively regulating anthocyanins biosynthesis; verify the shear action of VvmiR156 to VvSBP8, and transfer them together to tobacco leaves for transient expression in order to reveal the regulating effect of VvmiR156 on VvSBP8; analyze the preVvmiR156 promoter cis-element and expression level of transcription factors related with drought response in Vitis, screen the transcription factors related to drought response interacting with VvmiR156 promoter using yeast one-hybrid system (Y1H) and chromatin immunoprecipitation assay (ChIP), finally co-express the transcription factors and VvmiR156 promoter with LUC for confirming the signal transduction process of drought inducing the VvmiR156 expression. Overall, this program will reveal the molecular mechanism regulated water deficit promoting the anthocyanins biosynthesis during grape ripening.

花色苷是葡萄果实与葡萄酒中最主要的呈色物质，适度水分亏缺可促进葡萄果实花色苷积累，然而其机理尚不清楚。MicroR156是植物中一个非常保守的小分子非编码RNA。在证实水分亏缺时VvmiR156及其靶基因VvSBP8表达与花色苷合成有一定关系后，本项目将VvSBP8分别异源过表达红花烟草和病毒诱导基因沉默瞬时转化葡萄果皮，分析其对花色苷合成的负调控功能；利用5’RACE技术验证VvmiR156对VvSBP8的剪切作用，将二者共转化烟草叶片分析互作关系，揭示VvmiR156对VvSBP8表达的调控作用；分析VvmiR156启动子序列及葡萄干旱应答转录因子表达，并利用酵母单杂交、染色质免疫沉淀技术筛选与miR156启动子互作的转录因子，最后将筛选的转录因子与PVvmiR156-LUC瞬时共转烟草叶片，明确干旱诱导VvmiR156表达的信号传导过程，进而揭示水分亏缺诱导葡萄花色苷合成的作用机理。

干旱是制约葡萄与葡萄酒产业发展的重要因素之一。前期研究证实成熟期适度干旱可促进葡萄果实中花色苷的积累。本研究中，通过小RNAs组测序技术分析，共鉴定到158个已知的miRNAs和190个新的miRNAs，其中VvmiR156b和VvmiR156f等受水分亏缺诱导显著上调表达。多重比较分析表明，葡萄VvSBP8序列与VvmiR156成熟序列存在互补关系；水分亏缺处理葡萄后，VvSBP8与VvmiR156s的表达水平呈显著的时空差异，存在显著负相关关系；双荧光素酶试验结果显示VvmiR156能靶向剪切VvSBP8。过表达VvSBP8的离体果实花色苷积累降低，而基因沉默和基因干扰的葡萄果实颜色加深，花色苷积累增多，因此VvSBP8对花色苷积累具有负调控作用。qRT-PCR分析显示，VvSBP8的过表达降低了花色苷合成通路中转录因子VvMYB5b及结构基因VvUFGT等的表达水平。酵母单杂交试验表明，VvAREB2、VvCBF1和VvCBF3能够与VvmiR156家族启动子互作结合。双荧光素酶试验表明，VvAREB2、VvCBF1和VvCBF3能促进VvmiR156的表达。荧光素酶互补实验显示VvSBP8可抑制VvMYBA1与VvMYC1的互作。因此，干旱条件下，VvAREB2，VvCBF1和VvCBF3被诱导上调表达，之后结合在VvmiR156启动子上促进了VvmiR156表达，其靶基因VvSBP8转录被抑制，进而使VvSBP8对MBW复合体的干扰作用被削弱，进而促进了花色苷的积累。本项目的执行可为亏缺灌溉技术促进葡萄果实花色苷积累方面提供理论支撑，也为葡萄果实类黄酮次生代谢物合成调控提供新的认识。