川西獐牙菜SmSLS2基因的克隆、生物信息学及表达分析
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摘要
裂环马钱子苷合成酶(SLS)是环烯醚萜类化合物合成途径的限速酶。该研究根据川西獐牙菜转录组信息获得SmSLS2基因序列,对该基因进行克隆、生物信息分析、原核表达载体构建及蛋白体外诱导。结果表明SmSLS2 cDNA全长1 566 bp(GenBank编号MH535904),编码521个氨基酸,推测其蛋白相对分子质量为59. 79kDa,等电点为8. 92。结构域分析表明SmSLS2含一个跨膜结构域,SmSLS2的gDNA全长为2 576 bp,含5个外显子和4个内含子。SmSLS2蛋白与其他植物中SLS蛋白具有高度的相似性。进一步将SmSLS2构建到原核表达载体pET-28a-sumo上,转入大肠杆菌BL21(DE3)中,在37℃、0. 5 mmol·L~(-1)IPTG条件下进行原核表达,诱导出与预测蛋白大小一致的目的蛋白。该研究成功从川西獐牙菜中克隆了SmSLS2,为进一步阐明该基因在环烯醚萜合成途径中的作用和通过基因工程手段改良环烯醚萜类化合物产量提供了理论基础。
Secologanin synthase (SLS) is the key enzyme of secoiridoid pathway. We obtained the SmSLS2 gene full-length sequence according to the transcriptome of Swertia mussotii. We cloned the gene,analyzed the bioinformation of SmSLS2,constructed the vectors and performed the gene expression. The results showed that SmSLS2 cDNA complete sequence had a length of 1 566 bp (GenBank: MH535904),encoding 521 amino acid residues,and the pI of SmSLS2 was 8. 92. Domain analysis results showed that SmSLS2 had one transmembrane domain,and the protein secondary and tertiary structures were analyzed and forecasted. The gDNA sequence of SmSLS2 was 2 576 bp,contained five exons and four introns. The Sm SLS2 protein shared high identity with other SLS proteins of plants. Prokaryotic expression vector p ET-28 a-sumo-SmSLS2 was constructed and transformed into Escherichia coli BL21 (DE3) for expression under 37℃,and induced by 0. 5 mmol · L~(-1) IPTG. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size.Sm SLS2 gene was successfully cloned from S. mussotii,and it will provide a foundation for further functional research on Sm SLS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.
Secologanin synthase (SLS) is the key enzyme of secoiridoid pathway. We obtained the SmSLS2 gene full-length sequence according to the transcriptome of Swertia mussotii. We cloned the gene,analyzed the bioinformation of SmSLS2,constructed the vectors and performed the gene expression. The results showed that SmSLS2 cDNA complete sequence had a length of 1 566 bp (GenBank: MH535904),encoding 521 amino acid residues,and the pI of SmSLS2 was 8. 92. Domain analysis results showed that SmSLS2 had one transmembrane domain,and the protein secondary and tertiary structures were analyzed and forecasted. The gDNA sequence of SmSLS2 was 2 576 bp,contained five exons and four introns. The Sm SLS2 protein shared high identity with other SLS proteins of plants. Prokaryotic expression vector p ET-28 a-sumo-SmSLS2 was constructed and transformed into Escherichia coli BL21 (DE3) for expression under 37℃,and induced by 0. 5 mmol · L~(-1) IPTG. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size.Sm SLS2 gene was successfully cloned from S. mussotii,and it will provide a foundation for further functional research on Sm SLS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.
引文
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2.舒光明.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.Shu G M.Research progress on chemical constituents,pharmacological actions,and clinical applications of Swertia mussotii[J].Drugs&Clinic,2012,27(2):176-179.
3.郭爱华.龙胆科獐牙菜属药用植物化学成分和药理作用的研究进展[J].山西中医学院学报,2005,6(1):57-59.Guo A H.Research progress on chemical constituents and pharmacological actions of Swertia mussotii[J].Journal of Shanxi College of Traditional Chinese Medicine,2005,6(1):57-59.
4.罗翠婷,毛双双,陈河如,等.川西獐芽菜化学成分研究[J].中草药,2013,44(8):942-946.Luo C T,Mao S S,Chen H R,et al.Chemical constituents from Swertia mussotii[J].Chinese Traditional and Herbal Drugs,2013,44(8):942-946.
5.张建胜,王雪梅,董秀华,等.川西獐牙菜化学成分研究[J].中药材,2009,32(4):511-514.Zhang J S,Wang X M,Dong X H,et al.Studies on chemical constituents of Swertia mussotii[J].Journal of Chinese Medicinal Materials,2009,32(4):511-514.
6.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,et al.Research progress on chemical constituents,pharmacological actions,and clinical applications of Swertia mussotii[J].Drugs&Clinic,2012,27(2):176-179.
7.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):5-7.Zhu Y Y,Zhang X D,Zhang H Q.Research progress on pharmacological actions of Swertia mussotii[J].Chinese Wild Plant Resources,2008,27(1):5-7.
8.Wang J F,Zhao C Z,Liu C,et al.Introgression of Swertia mussotii gene into Bupleurum scorzonerifolium via somatic hybridization[J].BMC Plant Biology,2011,11:71.
9.Vaidya H,Goyal R K,Cheema S K.Anti-diabetic activity of swertiamarin is due to an active metabolite,gentianine,that upregulates PPAR-γgene expression in 3T3-L1 cells[J].Phytotherapy Research,2013,27(4):624-627.
10.朴金花,张杨,杨郁,等.胡黄连属植物环烯醚萜类化学成分和药理活性研究进展[J].中国实用医药,2007,2(34):142-144.Piao J H,Zhang Y,Yang Y,et al.Research progress on chemical constituents and pharmacological actions of Picrorhiza Royel[J].China Practical Medicine,2007,2(34):142-144.
11.贾诩,李茂星,陶锐,等.藏药独一味中的主要化学成分环烯醚萜苷化合物在动物的镇痛抗炎作用研究[J].中国临床药理学杂志,2015,31(8):635-639.Jia X,Li M X,Tao R,et al.Analgesic and anti-inflammatory activities of iridoid glycosides from Tibetan medicine Lamiophlomis rotata in Balb/C mice[J].The Chinese Journal of Clinical Pharmacology,2015,31(8):635-639.
12.匡雪君,王彩霞,邹立秋,等.长春花萜类吲哚生物碱生物合成与调控研究[J].中国中药杂志,2016,41(22):4129-4137.Kuang X J,Wang C X,Zou L Q,et al.Advance in biosynthesis of terpenoid indole alkaloids and its regulation in Catharanthus roseus[J].China Journal of Chinese Materia Medica,2016,41(22):4129-4137.
13.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 and Herbal Drugs,2017,48(5):962-970.
14.Xiang B B,Li X X,Wang Y,et al.Cloning and characterization of two iridoid synthase homologs from Swertia mussotii[J].Molecules,2017,22(8):E1387.
15.李晓雪,王勇,孙继奇,等.川西獐牙菜Sm DL7H基因原核表达及组织表达[J].西北植物学报,2018,38(8):1375-1381.Li X X,Wang Y,Sun J Q,et al.Prokaryotic expression and tissue specific expression of Sm DL7H in Swertia mussotii Franch[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(8):1375-1381.
16.Wang J F,Liu Y L,Cai Y F,et al.Cloning and functional analysis of geraniol 10-hydroxylase,a cytochrome P450from Swertia mussotii Franch[J].Bioscience,Biotechnology,and Biochemistry,2010,74(8):1583-1590.
17.Pandey S S,Singh S,Babu C S V,et al.Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis[J].Scientific Reports,2016,6:26583.
18.Liu Y,Wang H,Ye H C,et al.Advances in the plant isoprenoid biosynthesis pathway and its metabolic engineering[J].Journal of Integrative Plant Biology,2005,47(7):769-782.
19.DugéDe Bernonville T,Foureau E,Parage C,et al.Characterization of a second secologanin synthase isoform producing both secologanin and secoxyloganin allows enhanced de novo assembly of a Catharanthus roseus transcriptome[J].BMC Genomics,2015,16(1):619.
20.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].Scientific Reports,2017,7:43108.
21.Irmler S,Schr9der G,St-Pierre B,et al.Indole alkaloid biosynthesis in Catharanthus roseus:new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase[J].The Plant Journal,2000,24(6):797-804.
22.吴昕怡,刘小莉.环烯醚萜类成分生物合成途径及关键酶基因研究进展[J].中国民族民间医药,2017,26(8):44-48.Wu X Y,Liu X L.Progress of biosynthetic pathway and the key enzyme genes of iridoids[J].Chinese Journal of Ethnomedicine and Ethnopharmacy,2017,26(8):44-48.
23.韩梅,赵博,安志刚,等.长春花萜类吲哚生物碱生物合成途径中重要酶(DXR、SLS、G10H、STR)基因的克隆与表达[J].植物研究,2007,27(5):564-568.Han M,Zhao B,An Z G,et al.Cloning and expression of c DNA encoding key enzymes(DXR,SLS,G10H and STR)in terpene indole alkaloids biosynthesis pathway from Catharanthus roseus[J].Bulletin of Botanical Research,2007,27(5):564-568.
24.常贺.喜树SLS候选基因的分离及功能预测[D].哈尔滨:黑龙江大学,2009.Chang H.Cloning and function prediction of Camptothecia acuminate SLS gene[D].Harbin:Heilongjiang University,2009.
25.张晓东,李彩霞,王连春,等.滇龙胆Gr SLS1基因的克隆与原核表达[J].西北植物学报,2014,(7):1311-1317.Zhang X D,Li C X,Wang L C,et al.Cloning and prokaryotic expression of Gr SLS1 gene in Gentiana rigescens[J].Acta Botanica Boreali-Occidentalia Sinica,2014,34(7):1311-1317.
26.Hall T A.Bio Edit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J].Nucleic Acids Symposium Series,1999,41:95-98.
27.Tamura K,Peterson D,Peterson N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28(10):2731-2739.
28.卢晟晔,王丽颖.大肠杆菌中外源蛋白高效表达的影响因素及策略研究的新进展[J].中国实验诊断学,2006,10(9):1100-1103.Lu S Y,Wang L Y.New progress in the study of influencing factors and strategies for high-level expression of foreign protein in Escherichia coli[J].Chinese Journal of Laboratory Diagnosis,2006,10(9):1100-1103.
2.舒光明.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.Shu G M.Research progress on chemical constituents,pharmacological actions,and clinical applications of Swertia mussotii[J].Drugs&Clinic,2012,27(2):176-179.
3.郭爱华.龙胆科獐牙菜属药用植物化学成分和药理作用的研究进展[J].山西中医学院学报,2005,6(1):57-59.Guo A H.Research progress on chemical constituents and pharmacological actions of Swertia mussotii[J].Journal of Shanxi College of Traditional Chinese Medicine,2005,6(1):57-59.
4.罗翠婷,毛双双,陈河如,等.川西獐芽菜化学成分研究[J].中草药,2013,44(8):942-946.Luo C T,Mao S S,Chen H R,et al.Chemical constituents from Swertia mussotii[J].Chinese Traditional and Herbal Drugs,2013,44(8):942-946.
5.张建胜,王雪梅,董秀华,等.川西獐牙菜化学成分研究[J].中药材,2009,32(4):511-514.Zhang J S,Wang X M,Dong X H,et al.Studies on chemical constituents of Swertia mussotii[J].Journal of Chinese Medicinal Materials,2009,32(4):511-514.
6.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,et al.Research progress on chemical constituents,pharmacological actions,and clinical applications of Swertia mussotii[J].Drugs&Clinic,2012,27(2):176-179.
7.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):5-7.Zhu Y Y,Zhang X D,Zhang H Q.Research progress on pharmacological actions of Swertia mussotii[J].Chinese Wild Plant Resources,2008,27(1):5-7.
8.Wang J F,Zhao C Z,Liu C,et al.Introgression of Swertia mussotii gene into Bupleurum scorzonerifolium via somatic hybridization[J].BMC Plant Biology,2011,11:71.
9.Vaidya H,Goyal R K,Cheema S K.Anti-diabetic activity of swertiamarin is due to an active metabolite,gentianine,that upregulates PPAR-γgene expression in 3T3-L1 cells[J].Phytotherapy Research,2013,27(4):624-627.
10.朴金花,张杨,杨郁,等.胡黄连属植物环烯醚萜类化学成分和药理活性研究进展[J].中国实用医药,2007,2(34):142-144.Piao J H,Zhang Y,Yang Y,et al.Research progress on chemical constituents and pharmacological actions of Picrorhiza Royel[J].China Practical Medicine,2007,2(34):142-144.
11.贾诩,李茂星,陶锐,等.藏药独一味中的主要化学成分环烯醚萜苷化合物在动物的镇痛抗炎作用研究[J].中国临床药理学杂志,2015,31(8):635-639.Jia X,Li M X,Tao R,et al.Analgesic and anti-inflammatory activities of iridoid glycosides from Tibetan medicine Lamiophlomis rotata in Balb/C mice[J].The Chinese Journal of Clinical Pharmacology,2015,31(8):635-639.
12.匡雪君,王彩霞,邹立秋,等.长春花萜类吲哚生物碱生物合成与调控研究[J].中国中药杂志,2016,41(22):4129-4137.Kuang X J,Wang C X,Zou L Q,et al.Advance in biosynthesis of terpenoid indole alkaloids and its regulation in Catharanthus roseus[J].China Journal of Chinese Materia Medica,2016,41(22):4129-4137.
13.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 and Herbal Drugs,2017,48(5):962-970.
14.Xiang B B,Li X X,Wang Y,et al.Cloning and characterization of two iridoid synthase homologs from Swertia mussotii[J].Molecules,2017,22(8):E1387.
15.李晓雪,王勇,孙继奇,等.川西獐牙菜Sm DL7H基因原核表达及组织表达[J].西北植物学报,2018,38(8):1375-1381.Li X X,Wang Y,Sun J Q,et al.Prokaryotic expression and tissue specific expression of Sm DL7H in Swertia mussotii Franch[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(8):1375-1381.
16.Wang J F,Liu Y L,Cai Y F,et al.Cloning and functional analysis of geraniol 10-hydroxylase,a cytochrome P450from Swertia mussotii Franch[J].Bioscience,Biotechnology,and Biochemistry,2010,74(8):1583-1590.
17.Pandey S S,Singh S,Babu C S V,et al.Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis[J].Scientific Reports,2016,6:26583.
18.Liu Y,Wang H,Ye H C,et al.Advances in the plant isoprenoid biosynthesis pathway and its metabolic engineering[J].Journal of Integrative Plant Biology,2005,47(7):769-782.
19.DugéDe Bernonville T,Foureau E,Parage C,et al.Characterization of a second secologanin synthase isoform producing both secologanin and secoxyloganin allows enhanced de novo assembly of a Catharanthus roseus transcriptome[J].BMC Genomics,2015,16(1):619.
20.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].Scientific Reports,2017,7:43108.
21.Irmler S,Schr9der G,St-Pierre B,et al.Indole alkaloid biosynthesis in Catharanthus roseus:new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase[J].The Plant Journal,2000,24(6):797-804.
22.吴昕怡,刘小莉.环烯醚萜类成分生物合成途径及关键酶基因研究进展[J].中国民族民间医药,2017,26(8):44-48.Wu X Y,Liu X L.Progress of biosynthetic pathway and the key enzyme genes of iridoids[J].Chinese Journal of Ethnomedicine and Ethnopharmacy,2017,26(8):44-48.
23.韩梅,赵博,安志刚,等.长春花萜类吲哚生物碱生物合成途径中重要酶(DXR、SLS、G10H、STR)基因的克隆与表达[J].植物研究,2007,27(5):564-568.Han M,Zhao B,An Z G,et al.Cloning and expression of c DNA encoding key enzymes(DXR,SLS,G10H and STR)in terpene indole alkaloids biosynthesis pathway from Catharanthus roseus[J].Bulletin of Botanical Research,2007,27(5):564-568.
24.常贺.喜树SLS候选基因的分离及功能预测[D].哈尔滨:黑龙江大学,2009.Chang H.Cloning and function prediction of Camptothecia acuminate SLS gene[D].Harbin:Heilongjiang University,2009.
25.张晓东,李彩霞,王连春,等.滇龙胆Gr SLS1基因的克隆与原核表达[J].西北植物学报,2014,(7):1311-1317.Zhang X D,Li C X,Wang L C,et al.Cloning and prokaryotic expression of Gr SLS1 gene in Gentiana rigescens[J].Acta Botanica Boreali-Occidentalia Sinica,2014,34(7):1311-1317.
26.Hall T A.Bio Edit:a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[J].Nucleic Acids Symposium Series,1999,41:95-98.
27.Tamura K,Peterson D,Peterson N,et al.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J].Molecular Biology and Evolution,2011,28(10):2731-2739.
28.卢晟晔,王丽颖.大肠杆菌中外源蛋白高效表达的影响因素及策略研究的新进展[J].中国实验诊断学,2006,10(9):1100-1103.Lu S Y,Wang L Y.New progress in the study of influencing factors and strategies for high-level expression of foreign protein in Escherichia coli[J].Chinese Journal of Laboratory Diagnosis,2006,10(9):1100-1103.