茶树中两个ERF类转录因子的分离及不同茶树中温度胁迫的响应分析

刘志薇1,2, 熊洋洋3, 李彤1, 严雅君1, 韩洪润1, 吴致君1,2, 庄静1,2,*
南京农业大学1园艺学院, 2茶叶科学研究所, 3生命科学学院, 南京210095

通信作者:庄静;E-mail: zhuangjing@njau.edu.cn;Tel: 025-84395182

摘 要:

茶树作为我国重要的经济树种之一, 受温度胁迫的影响较大, ERF转录因子在调控温度胁迫过程中发挥重要的作用。本研究通过RT-PCR方法从茶树‘迎霜’叶片的cDNA中克隆得到2个ERF类转录因子基因, CsERF-B1CsERF-B4。序列分析表明, CsERF-B1CsERF-B4分别含有长为474和483 bp的开放阅读框, 编码157和160个氨基酸, 均含有AP2/ERF家族转录因子典型的保守AP2结合域。进化分析显示, 茶树CsERF-B1和CsERF-B4分别属于AP2/ERF家族ERF亚族的B1和B4组。通过比对发现, 这两个转录因子与其他物种ERF类蛋白的氨基酸序列具有很高的相似性, 如拟南芥、猕猴桃、杨树等。在三级结构上, CsERF-B1和CsERF-B4均有典型的1个α-螺旋和3个β-折叠结构。本文选取三种茶树材料‘迎霜’、‘安吉白茶’、‘云南十里香’进行不同温度胁迫下CsERF-B1CsERF-B4的响应分析。利用荧光定量PCR方法分析了CsERF-B1CsERF-B4基因分别在高温(38 ℃)和低温(4 ℃)处理0.5、1、2、4、8、12、24和48 h的表达情况。结果表明, CsERF-B1CsERF-B4基因均受高低温的诱导表达, 不同的茶树材料, CsERF-B1CsERF-B4基因表达有显著差异。同属ERF类转录因子的两个成员在三种具有抗寒性差异的茶树品种中的不同响应, 说明ERF转录因子在茶树温度胁迫调控中起重要作用。

关键词:茶树; 转录因子; ERF; 进化分析; 温度胁迫; 表达分析

收稿:2014-10-10   修定:2014-10-29

资助:国家自然科学基金项目(31200520)、江苏省自然科学基金项目(BK2012774)、教育部博士点基金项目(20120097120031)、博士后科学基金项目(2013M541686)和国家大学生创新创业训练计划(201410307030)。

Isolation and Expression Profiles Analysis of Two ERF Subfamily Transcription Factor Genes Under Temperature Stresses in Camellia sinensis

LIU Zhi-Wei1,2, XIONG Yang-Yang3, LI Tong1, YAN Ya-Jun1, HAN Hong-Run1, WU Zhi-Jun1,2, ZHUANG Jing1,2,*
1College of Horticulture, 2Tea Research Institute, 3College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, China

Corresponding author: ZHUANG Jing; E-mail: zhuangjing@njau.edu.cn; Tel: 025-84395182

Abstract:

Tea plant [Camellia sinensis (L.) O. Kuntze] is one of important economic tree species in our country. Throughout life, tea plants are challenged by various abiotic stresses, including temperature stress. In higher plants, ERF transcription factors provide important and effective signal regulation in defense temperature stress. In this study, the CsERF-B1 and CsERF-B4 genes, which encoding to the ERF transcription factor, were cloned by RT-PCR using cDNA as template, from Camellia sinensis cultivar ‘Yingshuang’. Sequence analysis indicated that the cDNA of CsERF-B1 and CsERF-B4 had a 474 and 483 bp open reading frame, encoding 157 and 160 amino acids, respectively. Both of the CsERF-B1 and CsERF-B4 contained a conserved AP2 domain, which is conserved in AP2/ERF family transcription factors. Phylogenetic analysis showed that CsERF-B1 and CsERF-B4 belong to B1 and B4 group of ERF subfamily of AP2/ERF family transcription factors. Based on sequence comparison, CsERF-B1 and CsERF-B4 had high homology in amino acid conserved regions with ERFs from other plants, such as Arabidopsis thaliana, Actinidia deliciosa, Populus trichocarpa, and so on. In the three-dimension structure, CsERF-B1 and CsERF-B4 had the typical structure with one α-helix and three β-strands. Three tea plant cultivars ‘Yingshuang’, ‘Anjibaicha’, and ‘Yunnanshilixiang’ were selected to analyze the expression profiles of CsERF-B1 and CsERF-B4 genes under the different temperature stresses. By quantitative real-time PCR, the expression profiles of the CsERF-B1 and CsERF-B4 genes were detected under high (38 ℃) and low (4 ℃) temperature stresses, at 0.5, 1, 2, 4, 8, 12, 24 and 48 h treatments, respectively. Under high or low temperature stresses, the expression levles of CsERF-B1 and CsERF-B4 genes were induced. The expression profiles showed difference among the three cultivars of tea plant. The different response of two transcription factors in three tea plant cultivars, belonging to ERF subfamily, indicated that ERF transcription factor may played important roles in regulation of temperature stress in tea plant.

Key words: Camellia sinensis; transcription factor; ERF subfamily; phylogenetic analysis; temperature stress; expression profile analysis

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