茶树AP2/ERF-B3类转录因子基因的克隆与表达特性分析

Isolation and expression profiles analysis of AP2/ERF-B3 group transcription factor from Camellia sinensis

植物在受到高温、低温、干旱和盐害等逆境胁迫时,ERF作为信号转导因子被诱导并调控其他抗逆基因的表达。研究茶树AP2/ERF-B3类转录因子在不同茶树品种间的分子特性、组织表达和逆境响应情况,将有助于了解AP2/ERF转录因子在茶树逆境调控中的作用。以2个茶树品种‘安吉白茶’和‘迎霜’为试验材料,通过RT-PCR方法分别从2种茶树的cDNA中克隆得到CsERF-B3基因。利用实时定量PCR技术检测该基因在茶树根、茎、叶、花各组织和4种非生物逆境胁迫处理(4℃低温、38℃高温、200 g·L-1PEG干旱处理、200 mmol·L-1NaCl)中的表达情况。结果表明:2种茶树中CsERF-B3基因全长均为639 bp,编码212个氨基酸,含有保守的AP2结合域,是植物典型的AP2/ERF家族转录因子;该转录因子属于AP2/ERF转录因子家族中的ERF亚族B3组;该转录因子是亲水性蛋白,无序化特征明显,并与拟南芥AtERF1具有相似的三级结构;该基因在茶树根中表达量最高,并且均能快速响应高温(38℃)、低温(4℃)和高盐(200 mmol·L-1NaCl)等非生物逆境胁迫。结论:环境中常见非生物胁迫可诱导茶树中CsERF-B3基因的表达,表明该AP2/ERF-B3类转录因子在茶树非生物胁迫中起着重要调节作用。

In higher plant,ERF subfamily transcription factor involved in growth and development,signal transduction,and regulated the expression of related genes in order to improve the resistance to the abiotic stresses,such as high temperature,low temperature,drought and salt damage.Here,the molecular characterization,tissue expression and abiotic stresses response of the AP2/ERF-B3 group transcription factor in different tea varieties were investigated.The results will contribute to understanding the roles of AP2/ERF-B3 transcription factor in tea plant（Camellia sinensis）. The CsERF-B3 genes,which encode to the AP2/ERF-B3 transcription factor,were cloned by RT-PCR method using cDNA as template,from two tea plant cultivars‘Anjibaicha＇and‘Yingshuang＇,respectively.Then,cDNA and deduced amino acid sequence,phylogenetic tree,physical and chemical characterization,hydrophilicity /hydrophobicity,molecular modeling and disordered residues were analyzed by related software.The expression profiles of the CsERF-B3 gene in the tissues of tea plants（roots,stems,leaves and flowers） were detected.The leaves,which were respectively treated with 2 h under 4 kinds of abiotic stresses（4 ℃ low temperature,38 ℃ high temperature,200 g·L-1PEG and 200 mmol·L-1NaCl）,were also detected by the quantitative real-time PCR analysis.Then,the multiple comparisons and plotting were finished by using Excel program.The results of sequence alignments and phylogenetic analysis showed that lengths of CsERF-B3 genes from the two tea plant cultivars were 639 bp,encoding 212 amino acids,respectively.The transcription factor of CsERF-B3 between 110- 145 amino acid sites contained the AP2 DNA binding domain where both contained a conservative WLG and YRG element.The CsERF-B3 of tea plants was a typical AP2/ERF family transcription factors in plants.Homologous phylogenetic tree showed that this ERF transcription factor was the closest relatives to AtERF1（AT4G17500.1） and AtERF2（AT5G47220.1） in Arabidopsis,they all belong to group B3,one group of the ERF subfamily transcription factors.The CsERF-B3 was hydrophilic protein and chemical properties were similar among related species.The CsERF-B3 and AtERF1 had similar three-dimension structure and disordered residues feature.The homologies of ABERF and YSERF were 92.1% and93.7% compared with the DNA binding domain of crystal structure PDB ID： 1gcc,respectively. The highest expression levels of theCsERF-B3 genes were found in the root of tea plant.The CsERF-B3 gene could respond quickly to high temperature（38 ℃）,low temperature（4 ℃） and high-salinity（200 mmol·L-1NaCl） treatments,respectively. The expression of CsERF-B3 gene was induced by abiotic stresses in tea plant,indicating that the transcription factor of AP2/ERF-B3 plays an important role in the regulation of abiotic stress in tea plant.