油菜BnICE1的克隆及表达分析

Cloning and Expression Analysis of a BnICE1 from Brassica napus L.

【目的】从超强抗寒油菜品种陇油6号中克隆抗寒转录因子BnICE1,并对其进行序列特征分析,研究BnICE1在低温胁迫下表达水平的变化,探讨BnICE1对油菜耐低温胁迫能力的影响。【方法】利用RACE技术获得油菜BnICE1全长的cDNA序列。对该序列进行生物信息学分析,采用实时荧光定量PCR研究BnICE1低温及不同组织表达量。【结果】cDNA片段全长1 737 bp,包含1 500 bp完整的开放阅读框,该基因编码499个氨基酸残基,分子量53.2 kD,等电点5.0。序列比对表明,该蛋白C端含有一个典型的bHLH结构域,与其它植物的ICE1蛋白具有较高的同源性,因此命名为BnICE1(GenBank登录号为JF268687)。进化树分析表明,BnlCE1同羊草和白菜亲缘关系较近。实时荧光定量PCR结果表明,该基因在油菜茎、叶和下胚轴均有表达,下胚轴中表达量最高,同时该基因的表达受低温胁迫诱导。【结论】从油菜中克隆了抗寒基因BnICE1,实时荧光定量PCR分析表明其在油菜适应低温胁迫的过程中发挥作用。

[Objective] To study the function of BnlCE1 in response to low temperature, transcription factor BnlCE1 which is involved in cold stress responsiveness in Brassica napus was cloned and its sequence was analyzed. [Method] The full-length eDNA of BnlCE1 was obtained by the technology of rapid amplification of eDNA ends （RACE）. The eDNA sequence and the deduced amino acid sequence were analyzed by bioinformatics method. The spatial-specific expression of BnICE1 in different tissues of the control plants and the expression patterns of BnlCE1 in leaves under cold stress condition were determined by real-time RT-PCR. [Result] In this study, a novel gene, BnlCEI, from B. napus was isolated and characterized. The full-length eDNA was 1 737 bp, containing a 1 500 bp opening reading frame（ORF）. The deduced protein was 499 amino acids with molecular weight 53.2 kD and isoelectric point 5.0. The sequence aligment demonstrated that the C-terminal of deduced BnlCEI protein exhibited a typical bHLH domain and was high identity with other ICE1 proteins from other species. So it was named the BnlCE1 （GenBank accession No. JF268687）. The result of phylogenetic tree showed that BnICE1 was closer to BclCE1 and BchlCE1. Analysis by real-time RT-PCR indicated that BnlCE1 was expressed in different tissues （stem, leaf, hypoeotyls） and that relatively higher abundance was observed in hypocotyls. [Conclusion] The transcription factor BnlCE1 was cloned from B. napus and the analysis by real-time RT-PCR suggested that BnICE1 played an important role in defending cold stress.