甘蔗S-腺苷蛋氨酸脱羧酶基因Sc-SAMDC的克隆和表达分析

【目的】克隆甘蔗(Saccharum officinarum)S-腺苷蛋氨酸脱羧酶(S-adenosylmethionine decarboxylase,SAMDC)基因,并进行序列特征、原核表达和不同逆境胁迫下表达特性分析。【方法】通过对甘蔗茎全长cDNA文库测序和分析,获得SAMDC基因cDNA全长,命名为Sc-SAMDC,并进行序列分析。随后将编码S-腺苷蛋氨酸脱羧酶的cDNA片段克隆到原核表达载体pET29a(+)中,构建融合表达质粒,转化至Esherichia coli BL21(DE3)中进行表达。最后利用定量PCR技术分析甘蔗幼苗中该基因在不同外源胁迫下的表达特性。【结果】序列分析显示,甘蔗Sc-SAMDC基因(GenBank Accession number:GQ246459)cDNA全长1968bp,存在3个读码框(袖珍读码框tORF、上游读码框uORF和主读码框mORF),mORF长1200bp,编码399个氨基酸的SAMDC酶原,预测分子量为43.6kD,该酶原含有两个高度保守的功能结构域(酶原剪切位点和PEST结构域)。原核表达产物经SDS-PAGE表明,Sc-SAMDC以融合蛋白形式表达,相对分子量约为50kD。定量PCR分析表明,Sc-SAMDC基因在聚乙二醇(PEG)、NaCl、水杨酸(SA)和H2O2外源胁迫下表达特性不同,受PEG和NaCl的诱导,受SA和H2O2的抑制。【结论】本研究成功地克隆了Sc-SAMDC基因并在原核生物中进行了表达研究,分析了该基因的表达特性,为其进一步的生物学功能研究及其应用奠定了基础。

Molecular Cloning of Sugarcane S-Adenosylmethionine Decarboxylase Gene (Sc-SAMDC) and Its Expression Analysis

Objective] In this study, the obtainment and sequence analysis of the S-adenosylmethionine decarboxylase (SAMDC) gene from sugarcane (Saccharum officinarum) was conducted, and then its expression in prokaryotic system and its expression profiles under different stress treatments were also carried out. Method] Firstly, through extensive sequencing and bioinformatics analysis, the full-length cDNA of sugarcane SAMDC gene, named as Sc-SAMDC, was obtained from sugarcane stem cDNA library, and its sequence characters were also analyzed. Secondly, the fused expression plasmid was constructed by inserting the cDNA fragment encoding the mature peptide of SAMDC into prokaryotic expression vector pET29a(+), and the positive recombinant was transformed into Esherichia coli BL21(DE3). Finally, the expression profiles of Sc-SAMDC in sugarcane seedling under different stresses were analyzed by Real-time qPCR. Result] Sequence analysis showed that the full-length cDNA sequence of Sc-SAMDC (GenBank Accession number: GQ246459) was 1 968 bp, with three open reading frames (ORFs), tiny ORF (tORF), upstream ORF (uORF) and main ORF(mORF). The mORF was 1 200 bp encoding 399 amino acid residues with a molecular mass of 43.6 kD, and the deduced protein had two highly conserved function domains (proenzyme cleavage site and PEST domain). The SDS-PAGE showed that the expression protein is a fusion protein and the molecular weight is about 50 kD. The result of Real time qPCR analysis showed that Sc-SAMDC responded differently to various exogenous treatments, induced by both PEG and NaCl, but inhibited by SA and H_2O_2. Conclusion] A SAMDC gene from sugarcane was successfully cloned and expressed in E. coli, the sequence characters and the expression profiles of this gene under different stresses were also analyzed.