川西獐牙菜SmDXS2基因的克隆及表达分析
|
摘要
1-脱氧-D-木酮糖-5-磷酸合成酶(1-deoxy-D-xylulose-5-phosphate synthase,DXS)是2-C-甲基-D-赤藻糖醇-4-磷酸(MEP)途径的第一个关键酶。该研究根据川西獐牙菜Swertia mussotii转录组数据,克隆获得其DXS2基因(Gen Bank注册号MH535905),命名为Sm DXS2。生物信息学分析表明Sm DXS2基因无内含子,其开放阅读框全长2 145 bp,编码714个氨基酸,分别属于20种氨基酸,其中丙氨酸、甘氨酸和色氨酸含量最高;相对分子质量为76. 91 k Da,理论等电点为6. 50,属亲水性蛋白;二级结构中α-螺旋占36. 83%,延伸链占9. 38%,loop占53. 78%;序列内部无信号肽,没有跨膜区,为非分泌型蛋白;序列中含有TPP结合位点、嘧啶结合结构域和C端的转酮酶结构域;系统发育分析结果显示Sm DXS2与滇龙胆DXS2共聚于一个分支。Sm DXS2基因在大肠杆菌中表达的重组蛋白相对分子质量约为94 k Da,与预测蛋白大小一致。该研究为进一步探讨该基因的功能和利用基因工程手段提高川西獐牙菜中环烯醚萜类化合物产量提供了理论依据。
1-deoxy-D-xylulose-5-phosphate synthase2(DXS2) is the first key enzyme of the MEP pathway,which plays an important role in terpene biosynthesis of plants. According to the data of Swertia mussotii transcriptome, DXS2 gene(Gen Bank number MH535905) was cloned and named as Sm DXS2. The bioinformatics results showed that Sm DXS2 has no intron,with a 2 145 bp open reading frame encoding a polypeptide of 714 amino acids. They are belonging to 20 kinds of amino acids,and the most abundant amino acids include Ala,Gly and Trp. The predicted protein molecular weight was 76. 91 k Da and its theoretical isoelectric point(p I) was6. 5,which belonging to a hydrophilic protein. α-Helix and loop were the major motifs of predicted secondary structure of DXS2. The three function domains are TPP_superfamily,Transket_pyr_ superfamily and Transketolase_C superfamily,respectively. The Sm DXS2 protein shared high identity with other DXS2 proteins of plants. Phylogenetic analysis showed that Sm DXS2 protein is grouped with the gentian DXS2 protein. The recombinant protein of Sm DXS2 gene in Escherichia coli was approximately 92. 00 k Da(containing sumo-His tag protein 13 k Da),which was consistent with the anticipated size.This work will provide a foundation for further functional research of Sm DXS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.
1-deoxy-D-xylulose-5-phosphate synthase2(DXS2) is the first key enzyme of the MEP pathway,which plays an important role in terpene biosynthesis of plants. According to the data of Swertia mussotii transcriptome, DXS2 gene(Gen Bank number MH535905) was cloned and named as Sm DXS2. The bioinformatics results showed that Sm DXS2 has no intron,with a 2 145 bp open reading frame encoding a polypeptide of 714 amino acids. They are belonging to 20 kinds of amino acids,and the most abundant amino acids include Ala,Gly and Trp. The predicted protein molecular weight was 76. 91 k Da and its theoretical isoelectric point(p I) was6. 5,which belonging to a hydrophilic protein. α-Helix and loop were the major motifs of predicted secondary structure of DXS2. The three function domains are TPP_superfamily,Transket_pyr_ superfamily and Transketolase_C superfamily,respectively. The Sm DXS2 protein shared high identity with other DXS2 proteins of plants. Phylogenetic analysis showed that Sm DXS2 protein is grouped with the gentian DXS2 protein. The recombinant protein of Sm DXS2 gene in Escherichia coli was approximately 92. 00 k Da(containing sumo-His tag protein 13 k Da),which was consistent with the anticipated size.This work will provide a foundation for further functional research of Sm DXS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.
引文
[1]宇妥·元丹贡布.四部医典[M].马世林等译.上海:上海科学技术出版社,1986:267.
[2]帝玛尔·丹增彭措.晶珠本草[M].毛继祖等译.上海:上海科学技术出版社,2012:125.
[3]朱钰叶,张喜德,张洪泉.藏药獐牙菜属植物的药理研究进展[J].中国野生植物资源,2008,27(1):5.
[4]薛长松,徐晶,李萃萍,等.环烯醚萜苷类化合物体内代谢及代谢物药理活性研究进展[J].中国中药杂志,2018,43(1):39.
[5] Hua W,Zheng P,He Y,et al. An insight into the genes involved in secoiridoid biosynthesis in Gentiana macrophylla by RNA-seq[J].Mol Biol Rep,2014,41(7):4817.
[6]张浩宇,樊俊苗,王婷,等.植物萜类合成关键基因DXS研究进展[J].生物技术通报,2018,34(3):1.
[7] Liu Y,Wang Y,Guo F X,et al. Deep sequencing and transcriptome analyses to identify genes involved in secoiridoid biosynthesis in the Tibetan medicinal plant Swertia mussotii[J].Sci Rep,2017,7:43108.
[8]张海晨,李彩晨,王元忠,等.滇龙胆1-脱氧-D-木酮糖5-磷酸合酶基因的克隆与表达分析[J].生物技术通报,2016,4:128.
[9] Chahed K,Oudin A,Guivarc HN,et al. 1-Deoxy-D-xylulose5-phosphate synthase from periwinkle. c DNA identification and induced gene expression in terpenoid indole alkaloid-producing cells[J]. Plant Physiol Biochem,2000,38(7/8):559.
[10] Sykora R,Legut D. Cloning and expression analysis of 1-deoxyD-xylulose-5-phosphate synthase gene from the medicinal plant Conyza blinii H. Lév[J]. Turk J Biol,2015,38(5):664.
[11]赵能,原晓龙,陈中华,等.滇牡丹1-脱氧-D-木酮糖-5-磷酸合酶(DXS)基因的克隆及功能分析[J].基因组学与应用生物学,2017,36(7):2919.
[12] Zhou W,Huang F,Li S,et al. Molecular cloning and characterization of two 1-deoxy-D-xylulose-5-phosphate synthase genes involved in tanshinone biosynthesis in Salvia miltiorrhiza[J]. Mol Breeding,2016,36(9):124.
[13] Hall T A. Bioedit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J]. Nucleic Acids Symposium Series,1999,41:95.
[14] Tamura K,Peterson D,Peterson N,et al. MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Mol biol evol,2011,28(10):2731.
[15]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.
[16]程波,杨伟俊,何江,等. 10种药用植物萜类合酶(DXS)生物信息学分析[J].中国农学通报,2015,31(2):146.
[17] Jadaun J S,Sangwan N S,Narnoliya L K,et al. Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera:active involvement of plastid isoprenogenic pathway in their biosynthesis[J]. Physiol Plant,2017,159(4):381.
[18]杨滢.植物萜类代谢途径关键酶的比较及其在丹参中的表达分析[D].西安:陕西师范大学,2012.
[19] Jibesh K P,Varun K,Hemant S,et al.Contents of therapeutic metabolites in Swertia chirayita correlate withthe expression profiles of multiple genes in corresponding biosynthesispathways[J]. Phytochemistry,2015,116:38.
[20] Sun R,Liu S,Gao J L,et al. Cloning and expression analysis of1-deoxy-D-xylulose-5-phosphate synthase gene from the medicinal plant Conyza blinii H. Lév[J]. Turk J Biol,2014,38(5):664.
[21] Duchêne E,Butterlin G,Claudel P,et al. A grapevine(Vitis vinifera L.)deoxy-D-xylulose synthase gene colocates with a major quantitative trait loci for terpenol content[J]. Theor Appl Genet,2009,118(3):541.
[22]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.
[23]田成旺,张铁军,蒋伶活.藏药川西獐牙菜的质量标准研究[J].中国实验方剂学杂志,2013,19(4):75.
[24]陈桂深,卢学峰,孙氰,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.
[2]帝玛尔·丹增彭措.晶珠本草[M].毛继祖等译.上海:上海科学技术出版社,2012:125.
[3]朱钰叶,张喜德,张洪泉.藏药獐牙菜属植物的药理研究进展[J].中国野生植物资源,2008,27(1):5.
[4]薛长松,徐晶,李萃萍,等.环烯醚萜苷类化合物体内代谢及代谢物药理活性研究进展[J].中国中药杂志,2018,43(1):39.
[5] Hua W,Zheng P,He Y,et al. An insight into the genes involved in secoiridoid biosynthesis in Gentiana macrophylla by RNA-seq[J].Mol Biol Rep,2014,41(7):4817.
[6]张浩宇,樊俊苗,王婷,等.植物萜类合成关键基因DXS研究进展[J].生物技术通报,2018,34(3):1.
[7] Liu Y,Wang Y,Guo F X,et al. Deep sequencing and transcriptome analyses to identify genes involved in secoiridoid biosynthesis in the Tibetan medicinal plant Swertia mussotii[J].Sci Rep,2017,7:43108.
[8]张海晨,李彩晨,王元忠,等.滇龙胆1-脱氧-D-木酮糖5-磷酸合酶基因的克隆与表达分析[J].生物技术通报,2016,4:128.
[9] Chahed K,Oudin A,Guivarc HN,et al. 1-Deoxy-D-xylulose5-phosphate synthase from periwinkle. c DNA identification and induced gene expression in terpenoid indole alkaloid-producing cells[J]. Plant Physiol Biochem,2000,38(7/8):559.
[10] Sykora R,Legut D. Cloning and expression analysis of 1-deoxyD-xylulose-5-phosphate synthase gene from the medicinal plant Conyza blinii H. Lév[J]. Turk J Biol,2015,38(5):664.
[11]赵能,原晓龙,陈中华,等.滇牡丹1-脱氧-D-木酮糖-5-磷酸合酶(DXS)基因的克隆及功能分析[J].基因组学与应用生物学,2017,36(7):2919.
[12] Zhou W,Huang F,Li S,et al. Molecular cloning and characterization of two 1-deoxy-D-xylulose-5-phosphate synthase genes involved in tanshinone biosynthesis in Salvia miltiorrhiza[J]. Mol Breeding,2016,36(9):124.
[13] Hall T A. Bioedit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J]. Nucleic Acids Symposium Series,1999,41:95.
[14] Tamura K,Peterson D,Peterson N,et al. MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Mol biol evol,2011,28(10):2731.
[15]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.
[16]程波,杨伟俊,何江,等. 10种药用植物萜类合酶(DXS)生物信息学分析[J].中国农学通报,2015,31(2):146.
[17] Jadaun J S,Sangwan N S,Narnoliya L K,et al. Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera:active involvement of plastid isoprenogenic pathway in their biosynthesis[J]. Physiol Plant,2017,159(4):381.
[18]杨滢.植物萜类代谢途径关键酶的比较及其在丹参中的表达分析[D].西安:陕西师范大学,2012.
[19] Jibesh K P,Varun K,Hemant S,et al.Contents of therapeutic metabolites in Swertia chirayita correlate withthe expression profiles of multiple genes in corresponding biosynthesispathways[J]. Phytochemistry,2015,116:38.
[20] Sun R,Liu S,Gao J L,et al. Cloning and expression analysis of1-deoxy-D-xylulose-5-phosphate synthase gene from the medicinal plant Conyza blinii H. Lév[J]. Turk J Biol,2014,38(5):664.
[21] Duchêne E,Butterlin G,Claudel P,et al. A grapevine(Vitis vinifera L.)deoxy-D-xylulose synthase gene colocates with a major quantitative trait loci for terpenol content[J]. Theor Appl Genet,2009,118(3):541.
[22]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.
[23]田成旺,张铁军,蒋伶活.藏药川西獐牙菜的质量标准研究[J].中国实验方剂学杂志,2013,19(4):75.
[24]陈桂深,卢学峰,孙氰,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.