刺葡萄VdERD6L15基因克隆及功能分析  

Cloning and functional analysis of the VdERD6L15 gene in Vitis davidii

作　　者：刘洁[1] 杨盛迪 曾雅婷 白描[1] 杨国顺[1] 李双江 LIU Jie;YANG Shengdi;ZENG Yating;BAI Miao;YANG Guoshun;LI Shuangjiang(College of Horticulture,Hunan Agricultural University/Hunan Engineering and Technology Research Center for Grapes,Changsha 410128,Hunan,China)

机构地区：[1]湖南农业大学园艺学院·湖南省葡萄工程技术研究中心,长沙410128

出　　处：《果树学报》2024年第9期1770-1780,共11页Journal of Fruit Science

基　　金：国家重点研发计划项目(2023YFD1200100);国家自然科学基金项目(32202531);国家现代农业产业技术体系项目(CARS-29-Zp-9)。

摘　　要：【目的】探究VdERD6L15在刺葡萄果实生长发育中的功能,从湘刺1号中克隆VdERD6L15基因及其启动子,进行基因功能研究和启动子活性分析。【方法】克隆VdERD6L15基因CDS序列,并对CDS进行生物信息学分析;构建VdERD6L15表达载体,并通过瞬时转化烟草确定VdERD6L15基因编码蛋白的亚细胞定位;同时,通过在己糖缺陷型酵母菌株EBY.VW4000中异源表达VdERD6L15探究其功能;此外,通过启动子截短试验确定VdERD6L15启动子的核心区域。【结果】成功克隆刺葡萄VdERD6L15基因编码序列,该编码序列长1461 bp,可编码486个氨基酸,具有膜蛋白特征,属于单糖转运蛋白家族。亚细胞定位试验确认VdERD6L15蛋白主要定位于液泡膜上。通过在酵母菌株EBY.VW4000中进行异源表达,验证了VdERD6L15具有转运葡萄糖的能力。利用PlantCARE数据库对VdERD6L15启动子顺式作用元件进行分析,发现其主要包含光响应元件、干旱胁迫和激素响应元件。通过GUS染色分析,进一步确定候选基因VdERD6L15启动子的关键调控区域位于-500 bp到-1000 bp之间。【结论】VdERD6L15是一个定位在液泡膜上的糖转运蛋白,具有转运葡萄糖的功能;VdERD6L15启动子的核心元件位于ATG上游500~1000 bp之间。【Objective】Vitis davidii Foëx.is a key wild grape germplasm resource in southern China,the sugars in the V.davidii berry are mainly accumulated in the form of glucose and fructose in the vacuole during the ripening period,and this process is mainly regulated by sugar transporter proteins.The Early-Response to Dehydration six-like(ERD6L)is a subfamily of Monosaccharide Transporters(MSTs),which has been proved as a key regulator in soluble sugar homeostasis.In this study,to investigate the role of VdERD6L15 gene in the growth and development of V.davidii berries,the coding sequence(CDS)and promoter of VdERD6L15 gene were cloned from Xiangci No.1,and its function and promoter activity were further analyzed.【Methods】The Pearson correlation coefficients and FDR values of the expression data of VdERD6LI5 gene and the soluble sugar content were calculated using the R packages GGally and ggplot2.The CDS of the VdERD6LI5 gene was cloned by reverse transcriptionpolymerase chain reaction(RT-PCR),and the sequence was biologically analyzed by ExPasY,TMHMM2.0,and SWISS-MODEL website.After constructing the VdERD6L15 related expression vector,tobacco was transformed to determine the subcellular localization of VdERD6L15,heterologous expression of VdERD6L15 was observed in yeast strain EBY.VW4000 to determine its function,and the core region of VdERD6L15 promoter was determined by GUS staining.【Results】Based on the Pearson correlation analysis of VdERD6L15 gene expression and sugar content,it was found that the expression of VdERD6LI5 was high in the early stage of berry development and low in the later stage,which was significantly and negatively correlated with the soluble sugar accumulation in the berry of V.davidii.The cDNA from the root tissues of V.davidii was used as a template,primers were designed by Oligo7 website,and the PCR amplification was performed.Sequence analysis showed that the length of the CDS of the VdERD6L15 gene was 1461 bp in length,encoding 486 amino acids and belonging to the Monosaccharide

关 键 词：刺葡萄 VdERD6L15 单糖转运蛋白 亚细胞定位 启动子活性 

分 类 号：S663.1[农业科学—果树学]