桑树转录因子CBF1基因的克隆及序列特征与低温应激表达

Molecular Cloning,Sequence Characteristics and Low Temperature Responsive Expression of Mulberry Transcription Factor CBF1 Gene

CBF(C-repeat binding factor)是一类与植物抗逆应答相关的转录因子。利用RT-PCR和RACE技术克隆桑树CBF1基因的全长cDNA序列,命名为MCBF1(GenBank登录号:JX186750)。该基因序列全长1 134 bp,包括693 bp的开放阅读框,87bp的5'端非翻译区和354 bp的3'端非翻译区;预测其编码的氨基酸序列具有保守的AP2结构域,且该结构域的两端拥有CBF家族特有的短多肽序列PKK/RPAGRxKFxETRHP和DSAWR,与其它植物CBF1的序列相似性较高;基于CBF1序列的系统进化树显示桑树与柑橘(Citrus jambhiri)、垂枝桦(Betula pendula)和切花月季(Rosa hybrid cultivar)的亲缘关系较近。将构建的pET28a-MCBF1原核表达载体转化大肠杆菌BL21后,诱导表达的MCBF1重组蛋白分子质量大小与预测值一致。半定量RT-PCR分析MCBF1基因在未经过低温处理的对照桑苗幼叶中无表达,而在低温处理后的桑苗幼叶中有表达,其中低温处理4 d后的表达量最高。克隆的MCBF1基因编码蛋白质具有典型的CBF家族结构域,并具有低温诱导表达的特点,推测其在桑树对低温的耐受性中发挥作用。

C-repeat binding factor （CBF） belongs to a family of transcription factors related to stress resistance and response of plant. In this experiment, we cloned the full length cDNA sequence of mulberry （Morus L. ） CBF1 transcription factor gene, designated as MCBF1 （GenBank accession number..JX186750）, by using RT-PCR and RACE techniques. This gene was 1 134 bp in full length, including an open reading frame of 693 bp, a 5＇-UTR （untranslated region） of 87 bp and a 3＇-UTR of 354 bp. Its deduced amino acid sequence was predicted to have the AP2 conservative domain, which is flanked by two short polypeptide sequences PKK/RPAGRxKFxETRHP and DSAWR unique to CBF family. Phylogenetic tree based on CBF1 amino acid sequences demonstrated that mulberry has close genetic relationship with Citrus jambhiri, Betula pendula and Rosa hybrid cultivar. After the constructed prokaryotic expression vector pET28a-MCBF1 was transformed into E. coil BL21 cells, the expressed recombinant protein had a molecular mass consistent with the predicted value. Semiquantitative RT-PCR analysis showed that MCBF1 gene was not expressed in young leaves of the control mulberry without low temperature treatment but expressed in young leaves of the mulberry with low temperature treatment. Its expression level was the highest in the mulberry under low temperature treatment for 4 d. Amino acid sequence encoded by MCBF1 gene has the typical domain of CBF family. Its expression can be induced by low temperature treatment. Therefore, it is related to tolerance of mulberry against low temperature stress.