梅花PmCBF基因的克隆及表达

Cloning and Expression of PmCBF Gene from Prunus mume

CBF转录因子在植物冷驯化中起着重要调控作用。以欧洲甜樱桃CBF基因为信息探针,从李属基因组数据库中得到一个候选序列,设计特异引物,通过PCR扩增和RT-PCR验证,发现与预测序列一致。该基因全长711bp,可编码225个氨基酸,序列分析发现其具有CBF基因的特征序列,包含保守的AP2/EREB DNA结合域以及CBF特征短多肽序列PKK/RPAGRxKFxETRHP和DSAWR。同源性分析显示,PmCBF和欧洲甜樱桃、矮扁桃CBF基因一致性较高,均在90%以上,与欧洲甜樱桃的氨基酸同源性达100%。实时荧光定量PCR分析发现:4℃低温胁迫下,PmCBF在转录水平的表达情况和其他植物CBF基因一致;4℃处理后PmCBF基因的表达开始上升,4 h时达最大,初步表明PmCBF在低温胁迫下上调表达。

The CBF transcription factor gene plays an important role in cold acclimation of plants.In this study,a putative sequences of CBF gene was identified in the Genome Datebase of Prunus by underlying the electronic cloning technique,using sequence of Prunus avim in formation.A cDNA clone and gene clone encoding CBF gene were isolated from the cDNA and gDNA prepared from leaf of Prunus mume by the gene specific RT-PCR and PCR amplification.The gene was 711 bp which are capable of encoding protein of 225 amino acids（aa）,respectively.Sequeces analysis showed that the PmCBF gene owns the characteristics of knowned CBF transcriptional factors,which contains an highly conserverd AP2/EREB DNA-binding domain and two special short amino acids sequences.Homology analysis indicate that the nucleotide sequences of the PmCBF gene were highly similar with those of Prunus avim,Prunus tella etc,both were up to 90% similarity.while the deduced amino acids sequences of it shared 100% identity with Prunus avim.The relative real-time PCR analysis showed that the expression of the PmCBF gene accord with other species after exposed to 4℃.The expression of the PmCBF gene increased at the beginning and arrived at the peak after 4 hours.So we can conclude that the PmCBF gene is induced by low temperature stress.