基于本实验室茶树品种迎霜的转录组数据,通过PCR方法从迎霜的DNA中克隆得到CsDREB-A4基因。分析显示,该基因含开放阅读框长708βbp,编码235个氨基酸,含有AP2/ERF家族转录因子典型的保守AP2结合域,并且与大豆、番茄、葡萄、拟南芥等的DREB转录因子有高度同源性。从进化树、亲/疏水性、无序化特性、二级和三级结构等方面进行预测与分析,结果表明,该转录因子属于AP2/ERF家族中的DREB亚族的A4组,大多数氨基酸属亲水性氨基酸,无序化特征比较明显,三级结构与AtERF1相似。利用实时荧光定量PCR分析表明,在迎霜、安吉白茶、云南十里香3个茶树品种中CsDREB-A4基因均受高温、低温诱导表达。在4℃处理下,在上述3个茶树品种中CsDREB-A4基因表达量均在24βh时达到最大值,分别为对照的23、4、43倍,并且CsDREB-A4基因在迎霜和云南十里香中均比安吉白茶的基因表达上调时间更长,上调程度更大。在38℃处理下,CsDREB-A4基因在迎霜和云南十里香中表达受抑制,均只在8βh时表达量高于对照,而在安吉白茶中表达量显著增加,在12βh时高达对照的2β720倍。
The CsDREB-A4 gene, which encoding to the DREB transcription factor, was cloned by PCR using DNA as template from tea plant (Camellia sinensis) cultivar ‘Yingshuang’, based on transcriptome data of ‘Yingshuang’. The open reading frame lengths of CsDREB-A4 gene was 708βbp, encoding 235 amino acids. The transcription factor of CsDREB-A4 contained the AP2 DNA binding domain, and had high homology with DERB transcription factors of Glycine max, Solanum lycopersicum, Vitis vinifera, Arabidopsis thaliana, and so on. Phylogenetic tree, hydrophilicity, disordered residues, two- and three-dimension structure of CsDREB-A4 transcription factor were also predicted and analyzed. Results showed that the disordered residues of CsDREB-A4 factor were obvious. The three-dimension structure of CsDREB-A4 factor was similar to AtERF1. The CsDREB-A4 factor, the majority of amino acid were hydrophilic, belonged to A4 group of DREB subfamily of AP2/ERF family transcription factors. The three-dimension structure was similar to AtERF1, the CsDREB-A4 gene was induced by high temperature and low temperature treatment in ‘Anjibaicha’, ‘Yingshuang’ and ‘Yunnanshilixiang’, respectively. The expression levels of CsDREB-A4 gene reached a maximum at 24 h in the three tea plant cultivars, which increased 23, 4, 43 times when exposed at 4℃. In ‘Yingshuang’ and ‘Yunnanshilixiang’, the expression levels of CsDREB-A4 gene were up-regulated more longer time and higher than that in ‘Anjibaicha’. The expression level of CsDREB-A4 gene was inhibited in ‘Yingshuang’ and ‘Yunnanshilixiang’ when exposed at 38℃ temperature, except at 8 h. In ‘Anjibaicha’, the expression level of CsDREB-A4 gene was increased obviously, and reached 2 720 times in 12 h than that in wild type tea plant.
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