川西獐牙菜SmDL7H基因原核表达及组织表达  

Prokaryotic Expression and Tissue-specific Expression of SmDL7 Hin Swertia mussotii Franch

作　　者：李晓雪[1] 王勇[1] 孙继奇 朱晔荣[1] 马琳[2] 向蓓蓓[2] LI Xiaoxue;WANG Yong;SUN Jiqi;ZHU Yerong;MA Lin;XIANG Beibei(College of Life Science,Nankai University,Tianjin 300071,China;School of Chinese Materia Medica,Tianjin University of Traditional Chinese Medicine,Tianjin 300193,China)

机构地区：[1]南开大学生命科学学院,天津300071 [2]天津中医药大学中药学院,天津300193

出　　处：《西北植物学报》2018年第8期1375-1381,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(81303303);天津市高等学校科技发展基金计划(20130203);天津市自然科学基金(18JCQNJC14000)

摘　　要：该研究根据川西獐牙菜转录组信息获得7-脱氧马钱子酸羟化酶(SmDL7H)基因的全长cDNA序列,对该基因进行同源克隆、生物信息学分析,并构建原核表达载体、转化大肠杆菌、进行原核表达和组织特异性表达分析,以探讨川西獐牙菜裂环烯醚萜合成途径中关键酶7-脱氧马钱子酸羟化酶(SmDL7H)基因的功能,为研究裂环烯醚萜类化合物合成途径奠定基础。结果表明:(1)成功克隆了川西獐牙菜SmDL7 H基因(GenBank登录号为MH243070);SmDL7 H基因开放阅读框为1 554bp,编码517个氨基酸,相对分子质量为59.5kD,等电点9.02;生物信息学预测SmDL7 H基因编码蛋白无信号肽。(2)多序列比对及进化树分析显示,SmDL7 H编码的蛋白与滇龙胆、长春花、金银花等植物的DL7 H基因编码的蛋白具有较高相似性。(3)将SmDL7 H基因连接到pET-28a原核表达载体,转化大肠杆菌Rosetta(DE3),用0.1mol/L IPTG于25℃诱导12h,原核表达分析发现在59.5kD处有目的蛋白出现,表明与之前预测的蛋白大小一致。(4)荧光定量PCR分析显示,SmDL7 H基因在川西獐牙菜叶、茎、花、根、愈伤组织中均有表达,其中在叶片中表达量最高,在根中表达量最低。In this research,according to the SmDL 7 H gene sequence of transcriptome of Swertia mussotii,we obtained the cDNA complete sequences by using method of homologous cloning,bioinformatics analysis,constructed prokaryotic expression vector,transformed into Escherichia coli and tissue-specific expression analysis,in order to identify the function of the 7-deoxyloganic acid-7-hydroxylase(SmDL7H)which is a key enzyme of the secoiridoid pathway in the S.mussotii.This work will also provide a foundation to study the secoiridoid pathway in S.mussotii.The results showed that:(1)SmDL 7 H gene from S.mussotii(GeneBank accesion number:MH243070)was cloned;SmDL 7 H gene open reading frame(ORF)of SmDL 7 H was 1 554 bp in length,which encoded 517 amino acids,with the isoelectric point of 9.02 and molecular mass of 59.5 kD;bioinformatics forecasted the SmDL7H protein with no signal peptide.(2)Phylogenetic analysis showed that SmDL7H protein shares high identity with Gentiana rigescens,Catharathus roseus,Cinchona calisaya and etc plants.(3)SmDL 7 H gene connected with expression vector pET-28a and then transformed into E.coil Rosetta(DE3)for heterologous expression.The recombinant protein was induced by 0.1 mol/L IPTG at 25℃for 12 h.Prokaryotic expression analysis showed that the expressed protein was 59.5 kD and accorded with our forecast.(4)qRT-PCR analysis indicated that SmDL 7 H was expressed in leaves,stems,flowers,roots and callus,and the highest in leaves,the lowest in roots.

关 键 词：川西獐牙菜 7-脱氧马钱子酸羟化酶 原核表达 组织表达分析 

分 类 号：Q785[生物学—分子生物学] Q786