PpWRKY4通过影响PpCCD4表达调控桃果实类胡萝卜素积累  

PpWRKY4 regulates carotenoids accumulation in peach fruit by affecting the expression of PpCCD4

作　　者：宋聪豪 姜超 金自清 张海朋 王小贝 侯楠 程钧 王伟 郑先波[1,2,3] 冯建灿 连晓东[1,2,3] 谭彬 SONG Conghao;JIANG Chao;JIN Ziqing;ZHANG Haipeng;WANG Xiaobei;HOU Nan;CHENG Jun;WANG Wei;ZHENG Xianbo;FENG Jiancan;LIAN Xiaodong;TAN Bin(College of Horticulture,Henan Agricultural University,Zhengzhou 450046,Henan,China;Henan Engineering and Technology Center for Peach Germplasm Innovation and Utilization,Zhengzhou,450046,Henan,China;Henan Provincial International Joint Laboratory of Horticultural Crops,Zhengzhou,450046,Henan,China)

机构地区：[1]河南农业大学园艺学院,郑州450046 [2]河南省桃种质资源创新与利用工程技术研究中心,郑州450046 [3]河南省园艺植物生物学国际联合实验室,郑州450046

出　　处：《果树学报》2024年第8期1504-1512,共9页Journal of Fruit Science

基　　金：河南省高等学校重点科研项目计划基础研究专项(24ZX010);河南省重点研发与推广专项(科技攻关)(232102110211);河南省高等学校重点科研项目(23A210023)。

摘　　要：【目的】克隆桃PpWRKY4基因,探究其在桃果实类胡萝卜素代谢过程中的调控机制。【方法】以白肉桃中油桃14号果实为材料,分析了其果实中类胡萝卜素含量变化趋势,克隆桃中PpWRKY4基因,对其进行生物信息学和表达分析。通过亚细胞定位、酵母单杂交技术和双荧光素酶试验确定PpWRKY4对PpCCD4的调控机制。【结果】随着中油桃14号果实成熟,PpCCD4基因表达量逐渐升高,类胡萝卜素含量(w,后同)由1.97μg·g^(-1)降至0.68μg·g^(-1)。PpWRKY4基因CDS区共1764 bp,编码587个氨基酸,蛋白分子质量为64.06 ku,等电点为6.00;通过氨基酸序列比对,其包含两个WRKY保守基序,为Ⅰ类WRKY蛋白。PpWRKY4基因在桃果实发育前期高表达,后期表达量较低,与PpCCD4表达趋势相反。亚细胞定位结果表明PpWRKY4定位在细胞核。酵母单杂交技术和LUC试验表明PpWRKY4通过结合PpCCD4启动子抑制其表达。【结论】PpWRKY4通过负调控PpCCD4表达进而调控桃果实类胡萝卜素降解,研究结果为进一步解析桃果实类胡萝卜素积累分子机制提供理论基础。【Objective】Peach(Prunus persica)is one of the most important economic fruit tree in the world.The color of fruit flesh is a notable feature for consumers and one of the important breeding objectives.The flesh color of white/yellow is a typical Mendelian trait controlled by a single locus(Y),and white phenotype(YY or Yy)is fully dominant over the yellow one(yy).The yellow-flesh peach mainly depends on the accumulation of carotenoids in chromoplasts.The carotenoids is considered as an indispensable part for human diet health and nutrition.The previous studies indicated that three types of the PpCCD4 mutation caused no expression or extremely lower expression of the PpCCD4 in yellowflesh peach,suggesting that the carotenoids would be negatively correlated with the expression of the PpCCD4.Although the PpCCD4 is considered as the key gene controlling the white/yellow flesh trait of peach,the regulatory mechanism of carotenoids degradation remains unclear.The PpWRKY4,a WRKY transcription factors associated with carotenoids degradation,was isolated from a white-flesh peach exhibiting lower carotenoids content in this study and the regulatory mechanism of the PpWRKY4 was explored in carotenoid metabolism of peach fruit.【Methods】The carotenoids content and the relative expression of the PpCCD4 at S1-S4 stages of the fruits of Zhongyoutao 14(CN14)were analyzed,respectively.The PpWRKY4(the homology of OfWRKY3)was cloned by PCR.PpWRKY4 and other three homologous proteins were analyzed for domains using MEGA 11.The expression pattern of the PpWRKY4 and PpCCD4 at S1-S4 periods of the fruits of CN14 were obtained by qRT-PCR.The cis-elements of the PpCCD4 promoter were analyzed using PlantRegMap and a binding site map was drawn by GSDS.The CDS of the PpWRKY4 was inserted into pSAK277-GFP vector,and then transformed into GV3101 and Marker mixed annotated Nicotiana benthamiana.The fluorescence in tobacco leaf cells was observed using laser scanning confocal microscopy after 36-48 h.The PpWRKY4 was cloned and inserted into

关 键 词：桃 WRKY转录因子 果肉颜色 类胡萝卜素 

分 类 号：S662.1[农业科学—果树学]