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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c DNA中克隆得到2个ERF类转录因子基因,Cs ERF-B1和Cs ERF-B4。序列分析表明,Cs ERF-B1和Cs ERF-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Cs ERF-B1和Cs ERF-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,Cs ERF-B1和Cs ERF-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Cs ERF-B1和Cs ERF-B4的响应分析。利用荧光定量PCR方法分析了Cs ERF-B1和Cs ERF-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,Cs ERF-B1和Cs ERF-B4基因均受高低温的诱导表达,不同的茶树材料,Cs ERF-B1和Cs ERF-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。
As one of the important economic tree species in China, tea tree is greatly affected by temperature stress. ERF transcription factor plays an important role in the regulation of temperature stress. In this study, two ERF-like transcription factor genes, Cs ERF-B1 and Cs ERF-B4, were cloned by RT-PCR from the cDNA of tea leaves. Sequence analysis showed that Cs ERF-B1 and Cs ERF-B4 contained open reading frames of 474 and 483 bp in length, encoding 157 and 160 amino acids, respectively, which contained the typical conserved AP2 binding domain of AP2 / ERF family transcription factor. Phylogenetic analysis showed that Cs ERF-B1 and Cs ERF-B4 belong to B1 and B4 subfamilies of AP2 / ERF family respectively. By comparison, the two transcription factors have high similarity with the amino acid sequences of ERF proteins of other species, such as Arabidopsis thaliana, kiwi, poplar and the like. On the tertiary structure, Cs ERF-B1 and Cs ERF-B4 have typical 1 α-helix and 3 β-sheet structures. In this paper, the responses of Cs ERF-B1 and Cs ERF-B4 under different temperature stress were selected from three kinds of tea materials ’Yingshuang’, ’Anji Baicha’ and ’Yunnan Shirley’. The expression of Cs ERF-B1 and Cs ERF-B4 genes at high temperature (38 ℃) and low temperature (4 ℃) for 0.5, 1, 2, 4, 8, 12, 24 and 48 h were analyzed by fluorescence quantitative PCR . The results showed that Cs ERF-B1 and Cs ERF-B4 genes were induced by high and low temperature, and the expression of Cs ERF-B1 and Cs ERF-B4 genes were significantly different among the different tea plant materials. The different responses of two members of the ERF-like transcription factor in three different tea cultivars with cold tolerance indicated that ERF transcription factor plays an important role in the regulation of tea plant stress.