川西獐牙菜SmDL7H基因原核表达及组织表达
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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 SmDL7H 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)SmDL7H gene fromS.mussotii(GeneBank accesion number:MH243070)was cloned;SmDL7H gene open reading frame(ORF)of SmDL7H 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 calisayaand etc plants.(3)SmDL7H gene connected with expression vector pET-28 aand then transformed into E.coil Rosetta(DE3)for heterologous expression.The recombinantprotein 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 SmDL7H was expressed in leaves,stems,flowers,roots and callus,and the highest in leaves,the lowest in roots.
In this research,according to the SmDL7H 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)SmDL7H gene fromS.mussotii(GeneBank accesion number:MH243070)was cloned;SmDL7H gene open reading frame(ORF)of SmDL7H 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 calisayaand etc plants.(3)SmDL7H gene connected with expression vector pET-28 aand then transformed into E.coil Rosetta(DE3)for heterologous expression.The recombinantprotein 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 SmDL7H was expressed in leaves,stems,flowers,roots and callus,and the highest in leaves,the lowest in roots.
引文
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[11]陈桂琛,卢学峰,孙菁,等.藏药川西獐牙菜的引种栽培研究[J].安徽农业科学,2005,33(2):272-273.CHEN G C,LU X F,SUN J,et al.Introduction and cultivation of Swertia mussotii Franch a famous Tibetan medicine[J].Journal of Anhui Agricultural Sciences,2005,33(2):272-273.
[12]向蓓蓓,李晓雪,王勇,等.川西獐牙菜牻牛儿基焦磷酸合成酶基因的克隆及表达分析[J].中草药,2017,48(5):962-970.XIANG B B,LI X X,WANG Y,et al.Cloning and expression of geranyl pyrophosphate synthase gene in Swertia mussotii[J].Chinese Traditional&Herbal Drugs,2017,48(5):962-970.
[13]XIANG B,LI X,WANG Y,et al.Cloning and characterization of two iridoid synthase homologs fromSwertia mussotii[J].Molecules,2017,22(8):1 387.
[14]YUE L,YI W,GUO F,et al.Deep sequencing and transcriptome analyses to identify genes involved in secoiridoid biosynthesis in the Tibetan medicinal plant Swertia mussotii[J].Scientific Reports,2017,7:43 108.
[15]赵忠娟,王燕飞,蔡云飞,等.4种高寒藏药材及其愈伤组织药效成分的高效液相色谱测定[J].山东大学学报(理学版),2010,45(1):41-45.ZHAO Z J,WANG Y F,CAI Y F,et al.Determination of efficacious components of gentianaceae plants and their calli by RP-HPLC[J].Journal of Shandong University,2010,45(1):41-45.
[2]井灵,张玉娟,陈志.川西獐牙菜的化学成分及药理研究进展[J].青海农林科技,2014,(4):55-57.JING L,ZHANG Y J,CHEN Z,et al.Research progress on chemical components and pharmacological actions of Swertia mussotii[J].Science&Technology of Qinghai Agriculture&Forestry,2014,(4):55-57.
[3]马徐,刘越,冯晓晓,等.藏茵陈川西獐牙菜的生物学研究进展[J].中华中医药杂志,2016,(3):948-950.MA X,LIU Y,FENG X X,et al.Biological research progress of Swertia mussotii Franch as a famous Tibetan medicine[J].China Journal of Traditional Chinese Medicine&Pharmacy,2016,(3):948-950.
[4]刘净,梁敬钰,谢韬.环烯醚萜类化合物近年研究进展[J].海峡药学,2004,16(1):14-19.LIU J,LIANG J Y,XIE T.Development of Iridoids in rencent years[J].Strait Pharmaceutical Journal,2004,16(1):14-19.
[5]董丽萍,倪梁红,赵志礼,等.龙胆属环烯醚萜类化学成分研究进展[J].中草药,2017,48(10):2 116-2 128.DONG L P,NI L H,ZHAO Z L,et al.Research progress on iridoids in Gentiana L[J].Chinese Traditional&Herbal Drugs,2017,48(10):2 116-2 128.
[6]罗玉燕,卢成瑛,陈功锡,等.裂环烯醚萜类化合物研究概况[J].食品科学,2010,31(21):431-436.LUO Y Y,LU C Y,CHEN G X,et al.Research progress in secoiridoid compounds[J].Food Science,2010,31(21):431-436.
[7]SALIM V,YU F,ALTAREJOS J,et al.Virus-induced gene silencing identifies Catharanthus roseus,7-deoxyloganic acid-7-hydroxylase,a step in iridoid and monoterpene indole alkaloid biosynthesis[J].Plant Journal,2013,76(5):754-765.
[8]KATANO N,YAMAMOTO H,IIO R,et al.7-Deoxyloganin 7-hydroxylase in Lonicera japonica cell cultures[J].Phytochemistry,2001,58(1):53-58.
[9]OUDIN A,COURTOIS M,RIDEAU M,et al.The iridoid pathway in Catharanthus roseus,alkaloid biosynthesis[J].Phytochemistry Reviews,2007,6(2-3):259-276.
[10]孟宪华,陈德道,张樱山,等.川西獐牙菜的化学成分、药理作用和临床应用研究进展[J].现代药物与临床,2012,27(2):176-179.MENG X H,CHEN D D,ZHANG Y S.Research progress on chemical constituents,pharmacological actions,and clinical applications of Swertia mussotii[J].Drugs&Clinic,2012,27(2):176-179.
[11]陈桂琛,卢学峰,孙菁,等.藏药川西獐牙菜的引种栽培研究[J].安徽农业科学,2005,33(2):272-273.CHEN G C,LU X F,SUN J,et al.Introduction and cultivation of Swertia mussotii Franch a famous Tibetan medicine[J].Journal of Anhui Agricultural Sciences,2005,33(2):272-273.
[12]向蓓蓓,李晓雪,王勇,等.川西獐牙菜牻牛儿基焦磷酸合成酶基因的克隆及表达分析[J].中草药,2017,48(5):962-970.XIANG B B,LI X X,WANG Y,et al.Cloning and expression of geranyl pyrophosphate synthase gene in Swertia mussotii[J].Chinese Traditional&Herbal Drugs,2017,48(5):962-970.
[13]XIANG B,LI X,WANG Y,et al.Cloning and characterization of two iridoid synthase homologs fromSwertia mussotii[J].Molecules,2017,22(8):1 387.
[14]YUE L,YI W,GUO F,et al.Deep sequencing and transcriptome analyses to identify genes involved in secoiridoid biosynthesis in the Tibetan medicinal plant Swertia mussotii[J].Scientific Reports,2017,7:43 108.
[15]赵忠娟,王燕飞,蔡云飞,等.4种高寒藏药材及其愈伤组织药效成分的高效液相色谱测定[J].山东大学学报(理学版),2010,45(1):41-45.ZHAO Z J,WANG Y F,CAI Y F,et al.Determination of efficacious components of gentianaceae plants and their calli by RP-HPLC[J].Journal of Shandong University,2010,45(1):41-45.