地黄RgGGPPS2基因克隆、生物信息学分析及表达分析  

Cloning, bioinformatic analysis and expression analysis of RgGGPPS2 gene from Rehmannia glutinosa

作　　者：赵乐[1,2] 马利刚[1,2] 俎梦航 冯卫生[1,2] 郑晓珂[1,2] 朱畇昊 

机构地区：[1]河南中医药大学药学院,河南郑州450046 [2]呼吸疾病诊疗与新药研发河南省协同创新中心,河南郑州450046

出　　处：《中草药》2017年第11期2269-2278,共10页Chinese Traditional and Herbal Drugs

基　　金：国家"十二五"科技支撑计划项目(2011BAI06B02);国家自然科学基金项目(81603232);河南省科技攻关计划项目(162102310468);河南中医学院博士科研基金(BSJJ2011-07;BSJJ2011-18)

摘　　要：目的为了研究地黄萜类化合物生物合成途径的功能基因,从地黄中克隆了一条新的地黄GGPPS基因(RgGGPPS2),进行生物信息学分析,原核表达、纯化以及基因表达模式分析。方法根据地黄转录组数据中RgGGPPS2基因的序列信息,设计特异性引物,克隆了RgGGPPS2基因的开放阅读框(ORF)序列,构建pET32a-RgGGPPS2原核表达载体,用IPTG在大肠杆菌BL21(DE3)中诱导表达RgGGPPS2重组蛋白,荧光定量PCR检测RgGGPPS2基因的组织特异性表达。结果 RgGGPPS2基因的ORF为867 bp,编码289个氨基酸。生物信息学分析结果发现RgGGPPS2蛋白含有2个富含天冬氨酸的基序FARM(first aspartate-rich motif)和SARM(second aspartate-rich motif),RgGGPPS2与芝麻、长春花等双子叶植物中GGPPS蛋白的同源性较高。通过构建pET-32a-RgGGPPS2原核表达载体在大肠杆菌中成功表达RgGGPPS2重组蛋白,利用Ni2+亲和色谱得到了纯化的RgGGPPS2重组蛋白。荧光定量PCR结果显示RgGGPPS2基因在根中表达量最高,其次是叶,茎中最低。结论克隆了RgGGPPS2基因,获得了纯化的RgGGPPS2重组蛋白,为研究RgGGPPS2基因在地黄萜类化合物生物合成途径中的功能奠定了基础。Objective In order to investigate the functional genes involved in terpenoids biosynthesis pathway of Rehmannia glutinosa, a new geranylgeranyl pyrophosphate synthase gene（RgGGPPS2） was isolated from R. glutinosa. Meanwhile, the bioinformatic analysis, prokaryotic expression, purification, and expression patterns of RgG GPPS2 gene were carried out. Methods Based on the transcriptome data of R. glutinosa, specific primers of RgG GPPS2 gene were designed, and an open reading frame（ORF） of RgG GPPS2 gene was isolated from R. glutinosa. By constructing the prokaryotic expression vector pE T32a-RgG GPPS2, the recombinant RgG GPPS2 protein was expressed in Escherichia coli BL21（DE3） cells under IPTG induction. The expression pattern of RgGGPPS2 in different tissues was detected by real-time PCR. Results RgG GPPS2 had an ORF of 867 bp, which encoded a protein of 289 amino acid residues. Bioinformatic analysis indicated that RgGGPPS2 protein contains the two conserved motifs FARM（first aspartate-rich motif, DDxxxxD） and SARM（second aspartate-rich motif, DDxx D）. Phylogenetic analysis revealed that RgG GPPS2 protein showed the highest homology with GGPPS protein from Sesamum indicum, Catharanthus roseus and other dicots. Through the construction of prokaryotic expression vector pE T-32a-RgG GPPS2, the recombinant RgG GPPS2 protein was successfully expressed in E. coli BL21（DE3） cells and the recombinant RgGGPPS2 protein was purified by Ni2＋ affinity chromatography. Real-time PCR analysis indicated that RgGGPPS2 was expressed in high transcript level in roots, lower level in leaves and the lowest level in stems.Conclusion The RgG GPPS2 gene was isolated from R. glutinosa and the recombinant RgG GPPS2 protein was obtained. The results of this study provide a foundation for functional characterization of RgG GPPS2 gene involved in terpenoids biosynthesis pathway of R. glutinosa.

关 键 词：地黄 RgGGPPS2 基因克隆 生物信息学分析 表达分析 

分 类 号：R282.12[医药卫生—中药学]