铁皮石斛蔗糖磷酸合成酶基因的克隆与原核表达

Molecular Cloning and Prokaryotic Expression of Sucrose Phosphate Synthase Gene from Dendrobium officinale

应用同源序列克隆法克隆了铁皮石斛蔗糖磷酸合成酶(SPS)基因cDNA全长,并进行了原核表达分析,为进一步研究该基因的时空表达、功能分析及多糖合成机理提供理论依据。结果表明:(1)铁皮石斛SPS基因cDNA全长3 502bp,编码区3 186bp,GenBank登录号JF423929。该基因编码1 061个氨基酸,与文心兰的SPS基因氨基酸序列的一致性最高为93%,与其他科植物SPS基因的氨基酸序列的一致性均高于60%。(2)原核诱导表达结果显示,SPS基因在大肠杆菌中的重组蛋白分子质量约为118.7kD,其表达与序列分析推测的结论一致。(3)生物信息学分析表明,铁皮石斛SPS基因的二级结构包括了螺旋、β-折叠和无规则卷曲,是非跨膜结构的亲水性不稳定蛋白,有2个功能结构域,分别是蔗糖合成功能域及糖基转移功能域。

This study used homologous sequence cloning method to clone the full length of sucrose phos phate synthase gene in Dendrobium officinale and induce prokaryotic expression. Results were as follows.- （1）The cDNA is 8 502 bp in length containing an open reading frame of 3 186 bp encoding 1 061 amino acids. The GenBank accession number is JF423929. On the amino acid sequence level, this gene shows the highest similarity （93%） with SPS gene of O. goldiana. The similarity of SPS gene were higher than 60% with other plants. （2）The gene was expressed in E. coli with molecular mass of 118.7 kD,which was con- sistent with the speculation of Biotech. （3） Secondary structure includes helix, beta-folding and coil, which is a hydrophilic, transmembrane and unstable proteins. There are two function structure domains which are sucrose synthesis functional domain and glyeosyl transfer function domain. This study provides the theory basis for research the gene spatio-temporal expression and function, polysaccharide synthesis mechanism and metabolism path in D. o f ficinale.