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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(Do HDS)的c DNA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Do HDS在不同组织的表达特征,构建原核表达载体p ET-28a(+)-Do HDS,转化大肠杆菌BL21(DE3)进行诱导表达。结果成功获得Do HDS的c DNA全长序列,Gen Bank登录号为KJ161312,全长2 666 bp,ORF为2 238 bp,编码745个氨基酸。Do HDS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(82 700)与理论值相符的融合蛋白。结论克隆了Do HDS的c DNA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Do HDS的功能提供了一定的理论基础。
Objective To clone 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase from Dendrobium officinale, HDS) gene in Escherichia coli to induce the fusion protein, and to explore the expression of the gene in different tissues of Dendrobium officinale. Methods The full-length cDNA of HDS (Do HDS) in D. officinale was obtained by RT-PCR and RACE methods. Bioinformatics analysis was carried out using related software and online websites. Real-time PCR was used to analyze the expression characteristics of Do HDS in different tissues. The prokaryotic expression vector p ET-28a (+) - Do HDS was constructed and transformed into E.coli BL21 (DE3) for expression. Results The complete cDNA sequence of Do HDS was successfully obtained. Gen Bank Accession No. KJ161312 was 2 666 bp in length with an ORF of 2 238 bp encoding 745 amino acids. Do HDS was expressed in all tissues and was the highest in stems, which was 5 times higher than that of protocorm. SDS-PAGE showed that the induced product was a fusion protein with the relative molecular mass (82 700) consistent with the theoretical value. Conclusion The full length cDNA of Do HDS was cloned and its expression pattern in different tissues was explored. The prokaryotic expression system was established and provided a theoretical basis for the subsequent study of the function of Do HDS.