FvEOBI转录因子调控草莓果实丁香酚代谢的分子机理

Eugenol is a volatile phenylpropanoid that contributes to flower and ripe fruit scent. The amount of this volatile is significantly lower in the fruits of the cultivated strawberry (Fragaria×ananassa) compared with the wild diploid ( Fragaria vesca). The regulatory networks governing eugenol biosynthesized is still unknown except eugenol metabolism regulated by EOBI, EOBII, MYB10 and ODO1. We have identified a ripening-related transcription R2R3-MYB transcription factor that seems to be the orthologous gene of PhEOBI from Petunia hybrida that contributes to the regulation of eugenol biosynthesis in petals, and named FvEOBI. In order to reveal the regulating molecular mechanisms of FvEOBI gene in the controlling of strawberry fruit eugenol biosynthesis, the wild diploid strawberry were selected as the test materials. Firstly, temporal expression of FvEOBI will be analysised, and its response to exogenous ABA treatment. Secondly, qPCR analysis of eugenol biosynthetic of altering FvEOBI over-expression, as well as the knock-down of this gene. Subsequently, promoters of FvEOBI and two key structural genes involved in eugenol production, CAD and EGS, will be cloned. EMSA, ChIP and LUC dual fluorescence, will be used to analyze if FvEOBI can transactive the CAD and EGS promoters in vivo and in vitro, respectively. Additionally, promoter of FvEOBI will be analyzed with EOBII and MYB10 proteins, to verify the specifically binding between R2R3-MYB transcriptional factors. Our study provides a valuable resource in regulating strawberry fruit volatile phenylpropanoid as well as the molecular-based breeding to improve the fruit quality.

苯环型挥发物丁香酚是草莓果实香气的重要组分，但多见于二倍体野生型草莓，而在八倍体栽培草莓中积累较少。已有研究表明，丁香酚代谢受EOBI、EOBII、MYB10和ODO1转录因子的调控，但具体的调控网络尚不完善；申请者先期从二倍体草莓中克隆得到一个MYB转录因子FvEOBI，并初步证实与果实丁香酚积累显著相关，但确切的分子机理尚未阐明。本项目拟以二倍体草莓“Hawaii4”为试材并做外源ABA处理，检测FvEOBI基因的时空表达模式；通过遗传转化分析该基因表达对丁香酚代谢的影响；克隆该基因及丁香酚合成关键酶基因CAD和EGS的启动子，采用EMSA、ChIP和LUC等方法，构建FvEOBI调控丁香酚代谢的上下游网络，明确EOBII、MYB10与FvEOBI之间的调控层级关系。本项目研究对于进一步明晰草莓果实丁香酚代谢的调控网络，以及提高栽培草莓果实的香气品质，都具有重要的理论意义和实用价值。

苯环型挥发物丁香酚是草莓果实香气的重要组分，但多见于二倍体野生型草莓，而在八倍体栽培草莓中积累较少。申请者前期通过测定50个草莓品种成熟期和6个国内广泛种植的草莓品种四个发育时期的香气成分并得到影响其分类的主要香气物质。从八倍体‘红颜’草莓中克隆得到一个新的MYB转录因子FaMYB63，亚细胞定位显示该转录因子位于细胞核中，qRT-PCR结果表明该基因时空表达模式与丁香酚含量呈正相关；通过瞬时注射草莓果实发现FaMYB63过表达和沉默使丁香酚代谢关键基因FaCAD1、FaEGS1和FaEGS2表达量显著上调或下调；克隆了FaMYB63以及丁香酚合成关键酶基因FaCAD1、FaEGS1和FaEGS2的启动子序列，采用EMSA、酵母单杂、GUS染色和LUC双荧光实验等方法验证了FaMYB63可以直接调控FaCAD1、FaEGS1和FaEGS2，并且FaMYB63直接调控另外两个丁香酚代谢相关的R2R3-MYB转录因子FaEOBII和FaMYB10，但FaMYB63并不受FaEOBII和FaMYB10的调控，明确了FaEOBII、FaMYB10和FaMYB63之间的调控关系。本项目对于进一步明晰草莓果实丁香酚代谢的分子调控网络，以及提高栽培草莓果实的香气品质，都具有重要的理论研究意义和实际应用价值。