地黄GGPPS1基因克隆及表达分析  

Clone and Expression Analysis of GGPPS1 Gene from Rehmannia glutinosa

作　　者：赵乐[1,2] 史晶晶[1,2] 马利刚[1,2] 何庆祥[1] 冯卫生[1,2] 郑晓珂[1,2] 朱畇昊 

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

出　　处：《西北植物学报》2016年第5期888-895,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家"十二五"科技支撑计划(2011BAI06B02);河南中医学院博士科研基金(BSJJ2011-07;BSJJ2011-18);第六届河南中医学院药学院大学生创新学习项目(YXCX[2015]3)

摘　　要：以地黄为材料,通过分析地黄转录组数据,设计特异性引物,克隆了地黄牻牛儿基牻牛儿基焦磷酸合酶(geranylgeranyl pyrophosphate synthase,GGPPS)基因的cDNA序列,命名为RgGGPPS1,GenBank登录号为KU258808。同时在生物信息学分析的基础上,进行原核表达、纯化以及组织特异性表达分析。结果显示:(1)RgGGPPS1基因开放阅读框为987bp,编码328个氨基酸。(2)生物信息学分析结果显示,RgGGPPS1蛋白含有2个富含天冬氨酸的基序(DDXXXXDD和DDXXD),与芝麻等双子叶植物中的GGPPS蛋白相似性较高。(3)利用构建的原核表达载体pET-32a-RgGGPPS1在大肠杆菌BL21(DE3)菌株中成功表达RgGGPPS1重组蛋白,采用Ni 2+亲和层析得到了纯化的RgGGPPS1重组蛋白。(4)荧光定量PCR结果显示,RgGGPPS1基因在根中表达量最高,叶、茎中表达量较低。研究结果为进一步研究RgGGPPS1基因在地黄环烯醚萜苷生物合成途径中的功能奠定了基础。Rehmannia glutinosa was used as experimental material in this study.Through analyzing the transcriptome data of R.glutinosaand designing specific primers,we isolated the cDNA sequence of geranylgeranyl pyrophosphate synthase（GGPPS）fromR.glutinosaand named as RgGGPPS1（GenBank accession No.KU258808）.Meanwhile,on the basis of bioinformatic analysis,we performed the prokaryotic expression,purification and tissue-specific expression analysis.The results indicated that：（1）RgGGPPS1has an open reading frame（ORF）of 987 bp,which encoded a protein of 328 amino acid residues.（2）Bioinformatic analysis indicated that RgGGPPS1 protein contains the two conserved motifs（DDXXXXDD）and（DDXXD）;RgGGPPS1protein showed the highest homology,92%identity,with GGPPS protein fromSesamum indicum.（3）By utilizing the construction of prokaryotic expression vector pET-32aRgGGPPS1,we successfully expressed the recombinant RgGGPPS1 protein in E.coli BL21（DE3）cells.Furthermore,the recombinant RgGGPPS1 protein was purified through Ni 2＋affinity chromatography.（4）Real-time PCR analysis indicated that RgGGPPS1 was expressed in high transcript level in roots,low levels in leaves and stems.The results of this study provided the fundamental information about RgGGPPS1 gene for follow-up research of its function involved in iridoid glycoside biosynthesis pathway.

关 键 词：地黄 RgGGPPS1基因 生物信息学分析 原核表达 表达分析 

分 类 号：Q785[生物学—分子生物学] Q786