铁皮石斛M型硫氧还蛋白基因的分子特征

Molecular Characterization of an M-type Thioredoxin Gene in Dendrobium officinale

利用RT-PCR和RACE技术,从珍稀濒危兰科药用植物铁皮石斛中分离到1个编码M型硫氧还蛋白(TRX)基因cDNA全长,命名为DoTRXm1(GenBank注册号KC178573)。序列分析结果表明,DoTRXm1基因长850bp,ORF(570bp)编码1条由189个氨基酸组成的肽链,分子量20.32kD,等电点9.44;DoTRXm1蛋白具有保守的硫氧还蛋白结构域(第87～187位氨基酸)和催化位点(106～124)。多序列比对和系统进化分析结果显示,DoTRXm1与植物M型TRXs基因有较高相似性(53%～70%),与水稻、玉米以及高粱等单子叶植物TRXs聚在1个分支,隶属于M型TRXs基因进化系统的Ⅱ类群。实时定量PCR分析显示,DoTRXm1基因为组成型表达,其转录本在石斛根中的相对表达量较高,为叶中的5.63倍,茎中次之(2.35倍),叶中最低。该研究为进一步解析DoTRXm1在铁皮石斛生长发育、逆境胁迫等过程中的生物学功能奠定基础。

In the present study,an M-type TRX gene,designated as DoTRXml （GenBank accession No. KC178573） ,was identified from a medicinally important orchid species Dendrobiurn officinale ,using RT PCR and RACE methods. Sequence analyses showed that the full length cDNA of DoTRXml was 850 bp and the 570 bp ORF encoded a 189 aa protein with a molecular weight of 20.32 kD and an isoelectric point of 9.44;The deduced DoTRXml protein contained a conserved therodoxin domain （87-187） and a catalyt- ic site （1064124）. Multiple sequence alignment demonstrated that DoTRXml was highly similar （53%-70%） to the M-type TRXs genes in plants. Phylogenetic analysis showed that DoTRXml was closely relat- ed to monocots including rice, maize, and sorghum, and belonged to the Ⅱ clade of plant M-type TRXs genes. Real time quantitative PCR （qRT-PCR） analysis revealed that DoTRXml was constitutively ex- pressed in leaves,stems,and roots of D. officinale. The transcription level of DoTRXml in roots was the highest （5.63 fold higher than that of leaves） ,followed by that of stems （2.35 fold） ,and leaves. Molecular characterization of DoTRXml development, stress responses functional elucidation of the gene involving in the