FaABCC1转运蛋白调控草莓果实细胞内花青素转运的分子机理

Anthocyanins are the main pigments contribute to the red or pink color of strawberry fruit. We previous found that the appropriate nitrogen deficiency could promote the coloration of strawberry fruit, and the metabolomics data indicated that ABC transporters were affected by the treatment. Using the Fragaria vesca genome sequence, members of ABC transporters were examined. We found one ABC transporter related to fruit coloration, and named FaABCC1. In order to reveal the molecular mechanisms of FaABCC1 gene in the regulation of strawberry fruit coloration, Fragaria × ananassa Duch. cv. ‘Benihoppe’ was selected as the test materials. Firstly, temporal expression and nitration treatment will be analyzed. Secondly, qPCR analysis of anthocyanin biosynthetic and regulator genes of altering FaABCC1 over-expression, as well as the knock-down of this gene. Finally, subcellular localization and yeast detection experiments were selected to determine the response of FaABCC1 to different cyanine glycosides specific (anthocyanidin-3-O-glucosyltransferase). Chromosome walking method will be used to clone the promoter, pFaABCC1::LUC/35S::REN will be co-transform into tobacco with or without MYB/bHLH to analyze if FaABCC1 is regulated by the anthocyanin transcription factors.Our study represents a valuable resource in anthocyanin transport in strawberry fruit coloration as well as the molecular-based breeding programmes to improve fruit quality.

花青素是形成草莓果实颜色的主要色素。前期研究发现，不同氮形态处理对草莓果实着色具有不同效应，且与ABC转运蛋白密切相关；在此基础上，申请者从草莓转色期果实中克隆得到一个ABC转运蛋白基因FaABCC1，并初步证实该基因与花青素积累和果实着色显著相关,但确切分子机理尚未阐明。本项目拟以‘红颜’草莓为试材，对FaABCC1基因进行时空表达模式研究以及氮处理响应分析；通过遗传转化草莓，分析FaABCC1基因表达水平对果实花青素积累等成熟相关基因表达的影响；采用亚细胞定位和酵母表达系统检测ABC转运活性，并确定FaABCC1转运花青素的具体种类；克隆该基因的启动子，通过启动子驱动LUC/REN双荧光标记与MYB/bHLH转录因子共转化烟草方法，分析相关转录因子对该基因的调控机制。本项目对于阐明FaABCC1转运蛋白调控花青素转运的分子机理，以及开展与草莓果实品质和成熟相关研究，都具有重要意义。

花青素是形成草莓果实颜色的主要色素。花青素在细胞质中合成，转运到相关细胞器中（如液泡）进行贮藏，以避免细胞结构受到破坏。前期研究结果发现ABC转运蛋白与草莓果实着色密切相关；在此基础上，申请者从草莓基因组中找到ABC类转运蛋白115个，进化树分析发现可以分为8个亚家族（6 个ABCA亚家族，29 个ABCB亚家族，16 个ABCC亚家族，1 个ABCD亚家族，2个 ABCE亚家族，5 个ABCF亚家族，46个 ABCG亚家族，10个 ABCI亚家族）。另外对ABC转运蛋白进行了基因结构域和蛋白结构分析。ABCC亚家族基因可能参与草莓果实着色过程，通过定量PCR实验检测了ABCC亚家族基因在草莓果实着色、种子着色和不同器官里面的表达模式。另外检测了ABCC亚家族基因对非生物胁迫和激素处理的响应模式，发现ABCC8 and ABCC11 基因在草莓果实着色过程中明显上调，且ABCC11基因受到Cd诱导表达。ABCC11 基因在草莓果实中沉默表达抑制了草莓果实着色，说明该基因是影响草莓果实着色的关键基因。ABCC11 基因在拟南芥中过量表达增强了对Cd的抗性。酵母突变体ycf转化证明了ABCC11基因具有专业Cd的功能。采用亚细胞定位结果显示ABCC11基因定位于液泡膜；克隆该基因的启动子，通过启动子驱动GUS标记与MYB/bHLH转录因子共转化烟草方法，发现该基因受到MYB10、bHLH3和MYC1基因的调控机制。除ABCC转运蛋白外，GST基因在草莓果实着色过程中也起到关键作用。本项目对于阐明ABCC转运蛋白调控花青素转运的分子机理，以及开展与草莓果实品质和成熟相关研究，都具有重要意义。