铁皮石斛DoSMT2基因的克隆与表达分析

Cloning and Expression Analysis of DoSMT2 Gene in Dendrobium officinale

摘要:

为了解SMT2基因在铁皮石斛（Dendrobium officinale）甾醇代谢过程中的作用，利用RACE技术克隆到1个DoSMT2基因，开放阅读框为1 089 bp，编码362个氨基酸，DoSMT2相对分子量为40.345 kD，理论等电点为8.13，属于稳定的亲水性蛋白。经BLAST P检索，DoSMT2蛋白属于AdoMet-MTases超级家族，含有4个S-腺苷蛋氨酸结合位点、1个甲基转移酶保守结构域和1个甾醇甲基转移酶C末端保守结构域。系统进化分析表明，DoSMT2与深圳拟兰（Apostasia shenzhenica）的SMT2亲缘关系最近，确定其属于SMT2家族。qRT-PCR分析结果表明，DoSMT2基因在茎和叶都能表达，10月份的表达量最高，叶片的表达量显著高于茎，推断叶片的甾醇代谢比茎活跃。构建了pET-29a-DoSMT2原核表达载体，并转化大肠杆菌BL21（DE3），IPTG诱导表达出预期大小的蛋白。这为铁皮石斛DoSMT2的甲基化机制及甾醇化合物代谢研究奠定基础。

Abstract:

In order to understand the function of SMT2 gene in sterol metabolism of Dendrobium officinale, DoSMT2 gene was cloned by RACE. The results showed that the full length of DoSMT2 gene was 1 446 bp with an ORF of 1 089 bp, encoding a protein of 362 amino acids. The DoSMT2 had relative molecular weight of 40.345 kD, and the theoretical isoelectric of 8.13, showing a stable hydrophilic protein. Retrieved by BLAST P, the DoSMT2 protein belonged to the AdoMet-MTases superfamily, containing four S-adenosylmethionine binding sites, one methyltransferase domain and one sterol methyltransferase C-terminal domain. The DoSMT2 was closely homologous to the SMT2 of Apostasia shenzhenica (PKA61629.1) by phylogenetic analysis, belonging to SMT2 family. The DoSMT2 gene was expressed in both stems and leaves by using qRT-PCR. The expression of DoSMT2 gene in October was the highest, and that in leaf was significantly higher than that in stem, it was suggested that sterol metabolism in leaves was more active than that in stems. The prokaryotic expression vector pet-29a-DoSMT2 was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant protein was expressed after IPTG induction. Therefore, these would provide the foundation for studies on the methylation mechanism of DoSMT2 and sterol metabolism of D. officinale.