桃类胡萝卜素合成关键基因PpCCD4的表达与启动子活性分析  

Expression and promoter activity analysis of PpCCD4 closely related to carotenoid synthesis in peach

作　　者：范家琪 吴金龙 李勇[1] 别航灵 王蛟[1] 郭健[1] 曹珂[1] 王力荣[1] FAN Jiaqi;WU Jinlong;LI Yong;BIE Hangling;WANG Jiao;GUO Jian;CAO Ke;WANG Lirong(Zhengzhou Fruit Research Institute,Chinese Academy of Agricultural Sciences,Zhengzhou 450009,Henan,China)

机构地区：[1]中国农业科学院郑州果树研究所,郑州450009

出　　处：《果树学报》2020年第9期1271-1280,共10页Journal of Fruit Science

基　　金：国家桃产业技术体系(CARS-30-1-04);中国农业科学院科技创新工程(CAAS-ASTIP-ZZFR-01)。

摘　　要：【目的】分析桃果实成熟过程中类胡萝卜素总量与关键基因PpCCD4表达的相关性,探究不同品种PpCCD4启动子的活性差异,进而解释PpCCD4在黄白肉果实中的表达差异。【方法】测定果实发育时期类胡萝卜素总含量,并与该时期PpCCD4的表达进行相关性分析,分别检测果实PpCCD4转录本差异,进行基因全长与启动子序列分析。构建PpCCD4启动子驱动GUS表达载体并转化农杆菌侵染桃果肉,根据果肉GUS染色分析2种启动子活性的差异,以期解释PpCCD4在果实发育时期表达量的差异。【结果】‘燕红’成熟过程中果实类胡萝卜素总含量降低,其PpCCD4在发育过程中的表达显著上调;‘金童6号’果实类胡萝卜素总含量升高,其PpCCD4的表达始终处于较低水平。‘金童6号’中PpCCD4相较‘燕红’在CDS区发生了2个碱基的插入,且两PpCCD4转录本不同,启动子克隆发现‘燕红’对应的启动子有较多的转录增强和启动元件,而黄肉品种则具有更多的抗逆应答元件;同时GUS染色结果表示,桃果肉中2种启动子驱动的GUS染色有明显的显色差异,启动子活性差异显著。【结论】黄肉品种中移码突变的产生造成了PpCCD4的功能丧失;2个品种果肉PpCCD4转录模式不同,其启动子活性的差异是造成PpCCD4在黄肉和白肉果实中表达差异显著的主要原因。【Objective】Carotenoids are important terpenoids in the secondary metabolites of plants.They not only have the function of protecting the plant itself by photoprotection and removal of reactive oxygen species,but also are important precursors of vitamins synthesized by the human body.Based on the color change of the yellow-flesh and the-flesh peach(Amygdalus persica L.)fruits during ripening,the correlation between the total carotenoids and the expression of the gene PpCCD4 was analyzed to explore the difference of the PpCCD4 promoter activity among different varieties,and then to explain the expression differences of the PpCCD4 in the yellow-flesh fruits.【Methods】The representative varieties of yellow flesh peach‘Golden Baby 6’and white flesh peach‘Yan Hong’were selected.The total carotenoid contents were measured at significant periods of fruit phenotypic change of the two cultivars,and specific primers were designed to analyze the fruit developmental period.Correlation analysis between the changes of total carotenoid contents and the expressions of the PpCCD4 in the flesh was made,specific primers were designed according to the peach reference genome.The differences of the PpCCD4 transcripts of the different peach varieties were detected.The sequences of PpCCD4 and promoter were cloned.The analysis of sequences and analysis of promoter regulatory elements was subsequently made.Using homologous recombination technology,the separately recovered promoter sequences were ligated with the digested pCAMBIA1301 vector plasmid to construct a PpCCD4 promoter to drive the GUS expression vector and to transform Agrobacterium to infect the peach pulp.The two promoters were analyzed according to the GUS staining of the pulp.The different activity of the promoters was employed to explain the difference of the expression of PpCCD4 during the fruit development.【Results】The total amount of carotenoids in the white flesh fruits during maturity was reduced to 6.6μg·g^-1,and the total amount of carotenoids in

关 键 词：桃 类胡萝卜素 PpCCD4 启动子活性 

分 类 号：S662.1[农业科学—果树学]