铁皮石斛S-腺苷酸脱羧酶基因DoSAMDC1的克隆及特征分析

Molecular cloning and characterization of S-adenosyl-L-methionine decarboxylase gene (DoSAMDC1) in Dendrobium officinale

S-腺苷甲硫氨酸脱羧酶(S-adenosyl-L-methionine decarboxylase,SAMDC)是多胺合成的关键酶,通过调节多胺的代谢途径参与植物的多项生理生化过程。本文利用cDNA末端快速克隆技术(RACE),首次从铁皮石斛共生萌发种子中分离得到一个新的SAMDC基因,命名为DoSAMDC1(GenBank注册号JX966243),并对其编码蛋白的理化性质、保守结构域等特征进行分析。生物信息学分析表明,DoSAMDC1基因的全长为1979bp,涵盖tiny-uORF、small-uORF和mainORF3个植物SAMDC基因特征ORF。mORF编码一条368个氨基酸的肽链,预测分子质量为40.7kD,等电点为5.2,编码蛋白不含信号肽,具有22个氨基酸的跨膜域(89～110位),具有植物SAMDC的典型结构特点,包括酶原剪切位点、PEST结构域及催化功能必须的氨基酸。序列比对及系统发育树分析结果表明DoSAMDC1与单子叶植物SAMDC具有很高的同源性,与双子叶植物的亲缘关系较远。应用实时荧光定量PCR对DoSAMDC1基因在石斛不同组织中的表达模式分析发现,该基因在未接菌的植物组织中表达变化差异不大,在接菌共生萌发的植物种子中显著上调表达,为未萌发种子的2.74倍,具有受真菌侵染诱导表达的特性,揭示其可能通过参与多胺调控途径在植物?真菌共生方面发挥作用。

S-Adenosyl-L-methionine decarboxylase （SAMDC） is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends （RACE）-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF （mORF）. The mORF was deduced to encode a 368 amino acid （aa） protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains （proenzyme cleavage site and PEST domain） and a 22-aa transmembrane domain （89-110）. Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutivelywithout significant change in the five tissues （not infected with fungi）. While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.