甜杨抗冻转录因子ICE1基因的in silico克隆及其分析

in silico Cloning and Analyzing of PsICE1 from Populus suaveolens,A Freezing-resistant Transcription Factor

ICE1基因编码类似MYC的bHLH转录因子,可特异地结合到CBF3启动子的MYC作用元件并诱导CBF/DREB1下游基因的转录表达。本文采用电子克隆的方法,以拟南芥ICE1蛋白序列为信息探针,利用杨树EST数据库和毛果杨基因组序列拼接的结果,设计引物并通过RT-PCR从甜杨克隆了杨树的第一个ICE1基因。其cDNA长1706bp,含有完整的开放阅读框,可编码543个氨基酸的MYC类蛋白。编码蛋白序列含有bHLH区,核定位信号(NLS)区,富S区和转膜区各1个。Blast分析表明,cDNA序列及其推导的氨基酸序列均与拟南芥和芥菜的ICE1存在着较高的同源性,说明所获得的cDNA可能是甜杨ICE1基因(PsICE1,DQ481236)。通过网络服务器平台进行PsICE1的功能预测,结果显示PsICE1含有bHLH保守功能域,具有多个磷酸化位点和跨膜区域。另外,ICE1的电子表达谱分析结果发现,ICE1几乎可在植物中整株表达,在多种组织和不同发育过程均表达,这也在一定程度上说明了ICE1是组成型表达,以及ICE1可能在植物的生长发育中也起着重要作用。

ICE1 encodes a MYC-like bHLH transcriptional activator, which could specially band to active domain of CBF3 promoter and induce transcriptal expression of cold-responsive genes downstream of CBF/DREB1. In this paper, we made the gene-specific primers based on the Populus EST information and P trichocarpa genomic sequences, as well as Arabidopsis ICE1 protein sequences, then we got the poplar ICE1 gene （GenBank Accession： DQ481236） from P.suaveolens by RT-PCR approach. The full length PsICE1 was 1 706 bp with a 1 629 bp open reading frame （ORF） and encoded a MYC-like protein of 543 amino acids. The predicted PsICE1 protein contained a nuclear localization signal （NLS）, a serine-rich region （S-rich）, a trasmembrane domain, N-glycosylation and kinase phosphorylation sites. Blast analysis revealed that PsICE1 was highly homologous to ICE1 from Arabidopsis thaliana and Capsella bursapastoris, which indicates that the cloned eDNA would be PsICE1 gene. Functional prediction in net server showed that PsICE1 had a potential basic helix-loop-helix （bHLH） with some phosphorylation sites and transmembrane domain. Moreover, electronic expression profiles revealed that ICE 1 could be expressed in all of plants and as well expressed at the different stages of growth and development, it might be obvious that ICE1 gene expression would be constitutive and it also might play an important role in the growth and development of plants.