茶树生物钟基因CsCCA1的鉴定及对光合特性的影响

刘文艳1,刘春方1,滕瑞敏1,杨妮1,王雅慧3,王广龙2,3,庄静1,*
1南京农业大学园艺学院, 茶叶科学研究所, 农业农村部华东地区园艺作物生物学与种质创制重点实验室, 南京 210095;2淮阴工学院生命科学与食品工程学院, 江苏淮安223003;3南京农业大学, 作物遗传与种质创新国家重点实验室, 南京210095

通信作者:庄静;E-mail: zhuangjing@njau.edu.cn

摘 要:

以茶树品种‘龙井43’为材料, 利用RT-PCR方法, 从茶树的cDNA中克隆得到茶树CsCCA1基因, 并对其进行生物信息学及表达量分析, 同时对不同时间点茶树叶片进行光合参数及气孔开度分析。结果显示,CsCCA1基因开放阅读框为1 626 bp, 共编码541个氨基酸。茶树CsCCA1蛋白序列与其他物种同源性序列最高达93.47%。CsCCA1蛋白与厚皮香、神秘果等物种亲缘关系相近, 与杜鹃、岩穗等物种亲缘关系相对较远。分析显示, CsCCA1是亲水性蛋白, 二级结构以无规则卷曲和延伸链为主要组成部分。根据蛋白互作网络分析, 茶树CsCCA1与LHY、TOC1核心元件作用较为直接。在24 h的光周期中, 净光合速率(Pn)呈现双峰型, 有光合“午休”现象, 胞间CO2浓度(Ci)、气孔导度(Gs)和蒸腾速率(Tr)在中午时均处于较高状态。气孔白天基本处于打开状态, 晚上处于闭合状态。实时荧光定量PCR结果显示, CsCCA1表达响应昼夜节律的变化, 在2~6 h CsCCA1表达量显著提高, 6~14 h呈显著下降趋势, 14~24 h表达量基本不变且较低。本试验结果为进一步研究CsCCA1基因对茶树生物钟的分子调控机制提供一定的参考。


关键词:茶树; 生物钟; CsCCA1基因; 表达分析; 光合特性

收稿:2021-10-28   修定:2021-12-30

资助:江苏省农业科技自主创新资金项目[CX(20)3114]、国家自然科学基金(31870681)和江苏高校优势学科建设项目(PAPD)。

Identification of circadian clock gene CsCCA1 and its effect on photosynthetic characteristics in tea plant

LIU Wenyan1, LIU Chunfang1, TENG Ruimin1, YANG Ni1, WANG Yahui3, WANG Guanglong2,3, ZHUANG Jing1,*
1Ministry of Agriculture and Rural Affairs Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Tea Research Institute, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China; 2School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian, Jiangsu 223003, China; 3State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China

Corresponding author: ZHUANG Jing; E-mail: zhuangjing@njau.edu.cn

Abstract:

The CsCCA1 gene was cloned from the cDNA of tea plant by RT-PCR with the tea cultivar ‘Longjing 43’ as the material, and the bioinformatics and expression analysis were carried out. Meanwhile, the photosynthetic parameters and stomata aperture of tea leaves at different time points were analyzed. The results showed that the open reading frame of CsCCA1 gene was 1 626 bp, encoding 541 amino acids. The homology of CsCCA1 protein sequence of tea plant with other species was up to 93.47%. The CsCCA1 protein was closely related to species such as Ternstroemia gymnanthera and Synsepalum dulcificum, and was relatively distant from species such as Rhododendron simsii and Galax urceolata. CsCCA1 is a hydrophilic protein. The secondary structure of tea CsCCA1 protein is composed of irregular curl and extended chain. According to the protein interaction network analysis in tea plants, CsCCA1 interacts directly with LHY and TOC1 core elements. During the 24 h photoperiod, the net photosynthetic rate (Pn) showed a double-peak pattern with a midday depression. The intercellular CO2 concentration (Ci), stomatal conductance (Gs) and transpiration rate (Tr) were high at noon. The stomata were basically open in the daytime, while closed at night. The results of real-time fluorescent quantitative PCR showed that the expression of CsCCA1 responded to the change of circadian rhythm. The expression level of CsCCA1 was significantly increased at 2–6 h, significantly decreased at 6–14 h, and basically unchanged at 14–24 h. The results of this study provide a reference for further studying the molecular regulation mechanism of CsCCA1 gene on circadian clock and biological rhythm in tea plant.


Key words: tea plant; circadian clock; CsCCA1 gene; expression analysis; photosynthetic characteristics

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