目的 从铁皮石斛Dendrobium officinale中克隆4-羟基-3-甲基-2-E-丁烯基-1-焦磷酸合成酶(4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase,HDS)基因,在大肠杆菌中诱导融合蛋白,并探究该基因在铁皮石斛不同组织中的表达规律。方法 采用RT-PCR和RACE等方法获取铁皮石斛HDS(DoHDS)的cDNA全长,利用相关软件和在线网站进行生物信息学分析,使用Real-Time PCR分析DoHDS在不同组织的表达特征,构建原核表达载体pET-28a(+)-DoHDS,转化大肠杆菌BL21(DE3)进行诱导表达。结果 成功获得DoHDS的cDNA全长序列,GenBank登录号为KJ161312,全长2666 bp,ORF为2238 bp,编码745个氨基酸。DoHDS在各组织中均有表达,在茎中表达量最高,为原球茎的5倍;SDS-PAGE结果显示诱导产物为1个相对分子质量(82700)与理论值相符的融合蛋白。结论 克隆了DoHDS的cDNA全长序列,探究其在不同组织中的表达模式,建立了原核表达体系,为后续研究DoHDS的功能提供了一定的理论基础。

Objective To clone the 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase(HDS) gene from Dendrobium officinale, induce fusion protein in Escherichia coli, and explore the regular pattern about HDS gene in different tissues of D.officinale.Methods RT-PCR and RACE technologies were used to clone the full-length cDNA of DoHDS.Using relevant softwares and online sites to analyze bioinformatics.Then the expression patterns of DoHDS were studied by real-time PCR.Constructing prokaryotic expression vector pET-28a(+)-DoHDS to induce the expression protein in E.coli BL21(DE3).Results The DoHDS gene was successfully obtained(GenBank accession number KJ161312), the full-length cDNA was 2666 bp and ORF was 2238 bp, coding the protein containing 745 amino acids.Relative real-time PCR analysis indicated that DoHDS showed the higher transcript abundance in the stems, 5 fold higher than protocorm-like bodies.The SDS-PAGE results showed that a relative molecular weight of 82700 recombinant protein was produced.Conclusion The cDNA encoding HDS from D.officinale is cloned.The prokaryotic expression vector and different tissues expression patterns of DoHDS are constructed.It is helpful for the future research on the mechanism of terpenoid biosynthesis in medicinal plants D.officinale.

安徽省教育厅自然科学重点项目(KJ2015A005);江淮地区大学农业科技服务模式关键技术集成与示范(2013BAD20B09)