菠萝MADS-box基因AcMADS2的克隆与表达分析

Cloning and expression analysis of MADS-box gene AcMADS2 from pineapple(Ananas comosus)

采用RT-PCR和RACE方法从菠萝克隆获得一个MADS-box基因,命名为Ac MADS2,Gen Bank登录号为KC257409。AcMADS2基因c DNA全长945 bp,ORF为687 bp,编码一个含有228个氨基酸的蛋白。序列分析和系统进化分析表明,AcMADS2具有保守的MADS-box结构域和半保守的K区,与其他植物中的AGL12亚家族MADS-box蛋白具有很高的同源性。生物信息学分析结果表明,Ac MADS2是不稳定的亲水蛋白,不具有跨膜结构和信号肽。α-螺旋、折叠延伸链及无规则卷曲构成了二级结构的主要蛋白框架。在三级结构中MADS-box结构域构成了该蛋白核心结构,并且作为转录因子可能定位于细胞核。实时荧光定量PCR分析表明,Ac MADS2基因在花期根中的表达量明显高于同一时期的小花、茎和叶;干旱和低温胁迫处理后,Ac MADS2基因在苗期根中的表达也明显增强。因此,推测Ac MADS2基因可能参与菠萝根发育的调控以及逆境胁迫反应。

A new cDNA sequence encoding MADS-box protein,designated as AcMADS2（Gen Bank accession number：KC257409）,from Ananas comosus was cloned by RT-PCR and RACE techniques.The full length cDNA of AcMADS2 has 945 bp nucleotides with an ORF of 687 bp encoding a precursor protein of 228 amino acid residues.Sequence analysis and evolution analysis indicated that the deduced protein contained conservative MADS-box domain and semi-conservative K domain,and shared highly homology to AGL12 clade of MADS-box proteins from other plants.Bioinformatics analysis results demonstrated that AcMADS2 was an unstable and hydrophilic protein without transmembrane region and signal peptide;α-helix,extended strand and random coil were the backbone of its secondary structure;the protein core structure with MADS-box domain in predictable protein 3D model;and might be located in the nucleus as a transcription factor.Real-time quantitative PCR analysis showed that the expression of AcMADS2 gene was much stronger in roots than in florets,stems and leaves at flowering stage,and the adversity stresses such as low temperature and drought could trigger a significant induction of AcMADS2 gene in roots at seedling stage.All these results suggested that the AcMADS2 protein may be involved in the regulation of root development,and play an important role in responses to adversity stresses.