美国白蜡抗寒转录因子FaCBF的克隆及表达分析

Cloning and Expression Analysis of Fa CBF from White Ash(Fraxinus americana)

为获得美国白蜡CBF基因的全长序列,根据不同物种CBF基因保守区域设计简并引物,利用RT-PCR和RACE技术进行试验。结果表明:其编码区为675bp,编码224个氨基酸。通过氨基酸序列同源比发现,美国白蜡CBF基因编码的氨基酸序列包含一个完整CBF蛋白的特征序列—AP2保守结构域,同时又存在单个氨基酸残基或基序的替换、插入和缺失;进化树分析表明:FaCBF与木本植物同处一个进化树分枝,其中,FaCBF基因与大叶钻天杨的CBF基因相似性最高;低温胁迫试验表明:FaCBF相对表达量随时间的延长而逐渐升高,在12h时,相对表达量达到最高,随后随时间的延长而逐渐降低。结果显示低温可以诱导FaCBF基因的表达,其可能在美国白蜡抗寒分子机制中发挥重要作用,为进一步分析FaCBF基因的功能和遗传转化奠定了基础。

With use of RT-PCR and rapid amplification of cDNA ends, a new full-length cDNAs of CBF gene was obtained from white ash, namely FaCBF. Sequence analysis showed that the open reading frame was 675 bp in length and encoded a polypeptide sequence of 224 amino acids, in which an AP2 DNA binding domain was found. The deduced amino acid sequence of FaCBF had high similarity to that of other reported CBF proteins. They were different each other by some substitutions,insertions and/or deletions involving single amino acid residues or motifs. Based on evolution analysis, it was clear that the genes from the same family were approximately clustered into a group, but the FaCBF was clustered into a separate group, indicating that there were some differences from other woody plants in evolution and the FaCBF from white ash was closely related to the CBF gene from Populus balsamifera. Real time quantitative RCR showed that expression of FaCBF was increased with the time extension and up to a maximum when cold induced for 12h, and then decreased. The results showed that low temperature stresses could induce the expression of FaCBF dramatically,it might play a key role during the development of cold resistant molecular mechanism in white ash, and the research provided the base for function analysis and genetic transformation of FaCBF gene.