梅花 CBF 转录因子的克隆及表达

摘要

根据GenBank中与梅花同属的桃、甜樱桃等已发表CBFs转录因子序列设计简并引物,采用PCR和RTPCR方法,从梅花基因组DNA和cDNA中克隆CBF转录因子片段.结果表明,两种途径获得的CBF基因序列一致,基因全长821 bp,编码238个氨基酸,其氨基酸序列具有典型的CBF蛋白特征,包含保守的AP2/EREB DNA 结合结构域及CBF家族蛋白特征短多肽序列(PKK/RPAGRxKFxETRHP和DSAWR).氨基酸相似性分析结果表明,该基因与欧洲甜樱桃、矮扁桃等CBF转录因子相似性较高.相对荧光定量PCR结果显示,4℃低温胁迫下,其表达量符合CBF转录因子表达特点,随着胁迫时间的增长表达量呈上升趋势,8h时达峰值,说明该基因在低温胁迫下上调表达.%Primers were designed according to the CBF transcription factors of Peach (Prunus persica), Sweet cherry (Prunus avium) et al from GenBank. Fragments of CBF gene were isolated from Prunus mume by PCR and RT-PCR, The CBF gene was 821 bp long, encoding a putative protein of 238 amino acids;The amino acids sequence owns the characteristics of the CBF protein,which contains an AP2/EREB DNA-binding domain and two special short amino acids sequences;Similarity analysis showed that the nu-cleotide were highly similarity to that of P. avium, P. tenella et al. Relative real-time PCR experiment showed the expression of PmCBFl was coincidence with the expression characteristics of the CBF gene after exposed to 4℃. The expression of PmCBFl increased at beginning and achieved the highest after 8 hour,indicating PmCBFl was induced under low temperature stress.