山葡萄VaCBF1转录因子基因的克隆与表达分析

Cloning and expression analysis of the transcription factor CBF gene from Vitis amurensis(VaCBF1)

【目的】克隆山葡萄(Vitis amurensis)CBF1转录因子基因(VaCBF1),为其功能的深入研究及其在植物抗寒基因工程中的应用奠定基础。【方法】根据植物CBF基因AP2/EREBP保守区设计1对简并引物,利用PCR法从山葡萄cDNA中扩增VaCBF1基因的中间片段。再根据中间片段区域设计2对特异引物,采用反向PCR法扩增VaCBF1基因的5′端和3′端序列。将中间片段与5′端和3′端序列拼接后得到山葡萄VaCBF1基因的cDNA全长序列,据此设计1对特异引物,PCR扩增VaCBF1基因编码区的全长序列,并对其进行生物信息学分析。同时,利用荧光定量PCR分析山葡萄VaCBF1基因在不同逆境胁迫(干旱、低温、盐胁迫)下的表达情况。【结果】成功地从山葡萄中克隆得到VaCBF1基因cDNA全长序列,其长度为762bp,编码253个氨基酸,在GenBank注册号为DQ517296。同源性分析证实,VaCBF1属于CBF转录因子家族。荧光定量PCR分析发现,低温胁迫可以诱导山葡萄VaCBF1基因高表达,而该基因的表达不受盐及干旱处理诱导。【结论】首次从山葡萄中克隆了VaCBF1基因,并证实该基因参与了植物对低温胁迫的应答。

【Objective】This study was conducted to clone and characterize the transcription factor CBF1 gene from Vitis amurensis （VaCBF1） for further functional studies and cold resistance application.【Method】A pair of degenerate primers were designed based on AP2/EREBP domain of VaCBF1 and a fragment of VaCBF1 was cloned from cDNA of V.amurensis using PCR.Then,two pairs of specific primers were designed based on middle fragment and the 5′ and 3′ end sequences of VaCBF1 gene were obtained by inverse PCR.A full sequence for cDNA of VaCBF1 was obtained by combining the middle fragment with sequences of 5′ and 3′.And a pair of primers was designed and used for cloning and analyzing the full length of VaCBF1 by PCR amplification and bioinformatics methods.The expression profiling of VaCBF1 gene exposed to various abiotic stresses （low temperature,drought and salt） was investigated using real time PCR as well.【Result】The full-length of VaCBF1 gene was 762 bp with 253 amino acids encoded （GenBank accession number DQ517296）.Phylogenetic analysis confirmed that VaCBF1 belonged to CBF transcription factor.Real time PCR results showed that expression of VaCBF1 was induced by low temperature treatment rather than drought and salt stresses.【Conclusion】This was the first clone and characterization of VaCBF1 gene from V.amurensis and it confirmed that VaCBF1 was related to the response to low temperature.