毛冬青三萜C-28位氧化修饰相关CYP450基因的发掘和功能鉴定
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摘要
药用植物毛冬青Ilex pubescens Hook.et Arn.含有大量的五环三萜及三萜皂苷,化学结构复杂多样。三萜类化合物骨架的修饰与其生物合成途径下游的一类关键酶——细胞色素P450单加氧酶(CYP450)密切相关。本研究通过分析毛冬青转录组数据,筛选到1个可能具有五环三萜C-28位氧化功能的CYP450基因(IpAO2)。克隆IpAO2基因并将其通过质粒转化法导入能高效合成amyrin的酿酒酵母工程菌中,GC-MS分析重组酵母代谢产物,发现IpAO2基因编码的蛋白可以将α-amyrin和β-amyrin分别氧化为熊果酸和齐墩果酸,确定IpAO2是一个五环三萜C-28位氧化酶基因。IpAO2基因的鉴定对全面阐明毛冬青三萜及三萜皂苷的生物合成机制具有重要意义,为后期开展毛冬青活性三萜类化合物的代谢工程研究奠定了基础。
The main components of the medicinal plant Ilex pubescens Hook.et Arn.are the pentacyclic triterpenoids with various chemical structures.The modification of the triterpenoid skeleton is closely related to a class of key enzymes downstream of the biosynthetic pathway,cytochrome P450 monooxygenase(CYP450).In this study,unigenes with a length of more than 1100 bp were retrieved from the previously obtained Ilex pubescens transcriptome data,and a cytochrome P450 gene(Unigene 0036170) proposed to have the oxidative function of pentacyclic triterpene C-28 was screened through phylogenetic tree analysis.The gene was named as IpAO2.The open reading frame of IpAO2 is 1443 bp,encoding 480 amino acids.RNA was extracted from the tender leaves of Ilex pubescens,and cDNA was obtained by reverse transcription using the RNA as the template.The specific primers AO2-F and AO2-R were designed according to the DNA sequence of IpAO2,and the IpAO2 gene was amplified by PCR using high fidelity enzyme.The IpAO2 gene was ligated into the pEASY cloning vector to obtain pEASY-IpAO2 plasmid.The ligation product was transformed into E.coli Trans1-T1 competent cells and positive clones were screened.The pESC-TRP plasmid was digested with restriction endonucleases to obtain a linearized vector.Primers V-AO2-F and V-AO2-R were designed and the DNA fragment was amplified by PCR using the pEASY-IpAO2 plasmid as template.The DNA fragment was ligated to the vector pESC-TRP to obtain pESC-TRP-IpAO2 recombinant plasmid.The pESC-TRP-IpAO2 plasmid was transformed into a strain of S.cerevisiae capable of efficiently synthesizing amyrin,and the expression of IpAO2 protein was induced with galactose.After extracting the total protein of recombinant S.cerevisiae,Western blot analysis was carried out through the 6×His antigen tag linked to the C-terminus of the target protein.The results showed that the recombinant protein was synthesized in accordance with the expected size.After 7 days of induction and cultivation,the cells of recombinant yeast were collected.The metabolites of cells were extracted and detected by GC-MS after silanization.Through GC-MS analysis of recombinant yeast metabolites,it was found that the protein encoded by the IpAO2 gene can oxidize α-amyrin and β-amyrin to ursolic acid and oleanolic acid,respectively,indicating that IpAO2 is a pentacyclic triterpene C-28 oxidase gene.Meanwhile,the transmembrane domain and protein tertiary structure of the IpAO2 were predicted using the Phyre2 online tool,the results showing that IpAO2 is a transmembrane protein.This study is of great significance to elucidate the biosynthetic mechanism of triterpenoids of Ilex pubescens,and lays a foundation for the further utilization of the metabolic engineering to produce triterpenoids of Ilex pubescens in S.cerevisiae.
The main components of the medicinal plant Ilex pubescens Hook.et Arn.are the pentacyclic triterpenoids with various chemical structures.The modification of the triterpenoid skeleton is closely related to a class of key enzymes downstream of the biosynthetic pathway,cytochrome P450 monooxygenase(CYP450).In this study,unigenes with a length of more than 1100 bp were retrieved from the previously obtained Ilex pubescens transcriptome data,and a cytochrome P450 gene(Unigene 0036170) proposed to have the oxidative function of pentacyclic triterpene C-28 was screened through phylogenetic tree analysis.The gene was named as IpAO2.The open reading frame of IpAO2 is 1443 bp,encoding 480 amino acids.RNA was extracted from the tender leaves of Ilex pubescens,and cDNA was obtained by reverse transcription using the RNA as the template.The specific primers AO2-F and AO2-R were designed according to the DNA sequence of IpAO2,and the IpAO2 gene was amplified by PCR using high fidelity enzyme.The IpAO2 gene was ligated into the pEASY cloning vector to obtain pEASY-IpAO2 plasmid.The ligation product was transformed into E.coli Trans1-T1 competent cells and positive clones were screened.The pESC-TRP plasmid was digested with restriction endonucleases to obtain a linearized vector.Primers V-AO2-F and V-AO2-R were designed and the DNA fragment was amplified by PCR using the pEASY-IpAO2 plasmid as template.The DNA fragment was ligated to the vector pESC-TRP to obtain pESC-TRP-IpAO2 recombinant plasmid.The pESC-TRP-IpAO2 plasmid was transformed into a strain of S.cerevisiae capable of efficiently synthesizing amyrin,and the expression of IpAO2 protein was induced with galactose.After extracting the total protein of recombinant S.cerevisiae,Western blot analysis was carried out through the 6×His antigen tag linked to the C-terminus of the target protein.The results showed that the recombinant protein was synthesized in accordance with the expected size.After 7 days of induction and cultivation,the cells of recombinant yeast were collected.The metabolites of cells were extracted and detected by GC-MS after silanization.Through GC-MS analysis of recombinant yeast metabolites,it was found that the protein encoded by the IpAO2 gene can oxidize α-amyrin and β-amyrin to ursolic acid and oleanolic acid,respectively,indicating that IpAO2 is a pentacyclic triterpene C-28 oxidase gene.Meanwhile,the transmembrane domain and protein tertiary structure of the IpAO2 were predicted using the Phyre2 online tool,the results showing that IpAO2 is a transmembrane protein.This study is of great significance to elucidate the biosynthetic mechanism of triterpenoids of Ilex pubescens,and lays a foundation for the further utilization of the metabolic engineering to produce triterpenoids of Ilex pubescens in S.cerevisiae.
引文
[1] Chinese Materia Medica Editorial Committee of the State Administration of Traditional Chinese Medicine,Chinese Materia Medica (中华本草)(Volume 5) [M].Shanghai:Shanghai Science and Technology Publishing House,1999:155.
[2] Cao LH,Miao MS,Xing WY,et al.Mechanism of total saponins from Radix Ilex Pubescens on improving cerebral ischemic tolerance in rats[J].Chin J Tradit Chin Med Pharm(中华中医药杂志),2017,32(12):5513-5517.
[3] Chen H,Sun F,Shao YB.Experimental study of Ilexonin A in preventing renal glomerulus of diabetic nephropathy rats[J].J New Chin Med(新中医),2014,46(1):171-174.
[4] Xiong TQ,Liao JY,Pang L,et al.Experimental study of effect of extract of Ilex pubescens on thrombosis and microcirculation[J].J Luzhou Med Coll(泸州医学院学报),2013,36(3):216-219.
[5] Wang ZR.Clinical observation of 143 cases of salpingitis obstruction treated mainly by Pubescent Holly Root,a Chinese herbal medicine for promoting blood circulation and removing blood stasis[J].Guangxi Med J(广西医学),1997,19(2):134.
[6] Xiong YX,Li C,Luo XT.Advances in chemical constituents and pharmacological activities of Ilex pubescens[J].J Chin Med Mater(中药材),2002,25(5):371-374.
[7] Jiang YP,Li H,Pan XZ,et al.Chemical constituents from the roots of Ilex pubescens[J].J Chin Med Mater(中药材),2013,36(11):1774-1778.
[8] Zhao ZX,Jin J,Lin ZZ,et al.Study on triperpenoid glycosides from roots of Ilex pubescens[J].China Pharm(中国药师),2011,14(5):599-601.
[9] Chen Y,Sun HY,Cao YP.Research progress on biosynthesis of triterpenoid saponins[J].Chin Wild Plant Resour(中国野生植物资源),2012,31(6):15-17.
[10] Bick J A,Lange B M.Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis:unidirectional transport of intermediates across the chloroplast envelope membrane[J].Arch Biochem Biophys,2003,415(2):146-154.
[11] Seki H,Tamura K,Muranaka T.P450s and UGTs:key players in the structural diversity of triterpenoid saponins[J].Plant Cell Physiol,2015,56(8):1463-1471.
[12] Xu HX,Li PJ,Liu W,et al.Research process of cytochrome P450 in organisms[J].J Agro-Environ Sci(农业环境科学学报),2002,21((2):188-191.
[13] Luo H,Sun C,Sun Y,et al.Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers[J].BMC Genomics,2011,12(Suppl 5):S5.
[14] Misra RC,Sharma S,Sandeep,et al.Two CYP716A subfamily cytochrome P450 monooxygenases of sweet basil play similar but nonredundant roles in ursane- and oleanane-type pentacyclic triterpene biosynthesis[J].New Phytol,2017,214(2):706-720.
[15] Augustin JM,Kuzina V,Andersen SB,et al.Molecular activities,biosynthesis and evolution of triterpenoid saponins[J].Phytochemistry,2011,72(6):435-457.
[16] Wen LL,Yun XY,Zheng XS,et al.Transcriptomic comparison reveals candidate genes for triterpenoid biosynthesis in two closely related Ilex species[J].Front Plant Sci,2017,8:634.
[17] Qin JL,Yun XY,Ji XY,et al.Identification and functional analysis of a C-28 oxidase gene involved in the biosynthesis of triterpenoid saponins from Ilex pubescens[J].Lishizhen Med Mat Med Res(时珍国医国药),2019,30(2):303-307.
[18] Zhao WZ,Ju WZ,Tan HS.Research Progress of triterpenoid saponin in pubescent holly root[J].Acta Chin Med(中医学报),2015,30(8):1181-1184.
[19] Huang LL,Li J,Ye HC,et al.Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus[J].Planta,2012,236(5):1571-1581.
[20] Moses T,Pollier J,Faizal A,et al.Unravelling the triterpenoid saponin biosynthesis of the African Shrub Maesa lanceolata[J].Mol Plant,2015,8(1):122-135.
[21] Kim O T,Um Y,Jin ML,et al.A novel multifunctional C-23 oxidase,CYP714E19,is involved in asiaticoside biosynthesis[J].Plant Cell Physiol,2018,59(6):1200-1213.
[22] Carelli M,Biazzi E,Panara F,et al.Medicago truncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins[J].Plant Cell,2011,23(8):3070-3081.
[23] Han J,Kim M,Ban Y,et al.The involvement of beta-amyrin 28-oxidase (CYP716A52v2) in oleanane-type ginsenoside biosynthesis in Panax ginseng[J].Plant Cell Physiol,2013,54(12):2034-2046.
[2] Cao LH,Miao MS,Xing WY,et al.Mechanism of total saponins from Radix Ilex Pubescens on improving cerebral ischemic tolerance in rats[J].Chin J Tradit Chin Med Pharm(中华中医药杂志),2017,32(12):5513-5517.
[3] Chen H,Sun F,Shao YB.Experimental study of Ilexonin A in preventing renal glomerulus of diabetic nephropathy rats[J].J New Chin Med(新中医),2014,46(1):171-174.
[4] Xiong TQ,Liao JY,Pang L,et al.Experimental study of effect of extract of Ilex pubescens on thrombosis and microcirculation[J].J Luzhou Med Coll(泸州医学院学报),2013,36(3):216-219.
[5] Wang ZR.Clinical observation of 143 cases of salpingitis obstruction treated mainly by Pubescent Holly Root,a Chinese herbal medicine for promoting blood circulation and removing blood stasis[J].Guangxi Med J(广西医学),1997,19(2):134.
[6] Xiong YX,Li C,Luo XT.Advances in chemical constituents and pharmacological activities of Ilex pubescens[J].J Chin Med Mater(中药材),2002,25(5):371-374.
[7] Jiang YP,Li H,Pan XZ,et al.Chemical constituents from the roots of Ilex pubescens[J].J Chin Med Mater(中药材),2013,36(11):1774-1778.
[8] Zhao ZX,Jin J,Lin ZZ,et al.Study on triperpenoid glycosides from roots of Ilex pubescens[J].China Pharm(中国药师),2011,14(5):599-601.
[9] Chen Y,Sun HY,Cao YP.Research progress on biosynthesis of triterpenoid saponins[J].Chin Wild Plant Resour(中国野生植物资源),2012,31(6):15-17.
[10] Bick J A,Lange B M.Metabolic cross talk between cytosolic and plastidial pathways of isoprenoid biosynthesis:unidirectional transport of intermediates across the chloroplast envelope membrane[J].Arch Biochem Biophys,2003,415(2):146-154.
[11] Seki H,Tamura K,Muranaka T.P450s and UGTs:key players in the structural diversity of triterpenoid saponins[J].Plant Cell Physiol,2015,56(8):1463-1471.
[12] Xu HX,Li PJ,Liu W,et al.Research process of cytochrome P450 in organisms[J].J Agro-Environ Sci(农业环境科学学报),2002,21((2):188-191.
[13] Luo H,Sun C,Sun Y,et al.Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers[J].BMC Genomics,2011,12(Suppl 5):S5.
[14] Misra RC,Sharma S,Sandeep,et al.Two CYP716A subfamily cytochrome P450 monooxygenases of sweet basil play similar but nonredundant roles in ursane- and oleanane-type pentacyclic triterpene biosynthesis[J].New Phytol,2017,214(2):706-720.
[15] Augustin JM,Kuzina V,Andersen SB,et al.Molecular activities,biosynthesis and evolution of triterpenoid saponins[J].Phytochemistry,2011,72(6):435-457.
[16] Wen LL,Yun XY,Zheng XS,et al.Transcriptomic comparison reveals candidate genes for triterpenoid biosynthesis in two closely related Ilex species[J].Front Plant Sci,2017,8:634.
[17] Qin JL,Yun XY,Ji XY,et al.Identification and functional analysis of a C-28 oxidase gene involved in the biosynthesis of triterpenoid saponins from Ilex pubescens[J].Lishizhen Med Mat Med Res(时珍国医国药),2019,30(2):303-307.
[18] Zhao WZ,Ju WZ,Tan HS.Research Progress of triterpenoid saponin in pubescent holly root[J].Acta Chin Med(中医学报),2015,30(8):1181-1184.
[19] Huang LL,Li J,Ye HC,et al.Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus[J].Planta,2012,236(5):1571-1581.
[20] Moses T,Pollier J,Faizal A,et al.Unravelling the triterpenoid saponin biosynthesis of the African Shrub Maesa lanceolata[J].Mol Plant,2015,8(1):122-135.
[21] Kim O T,Um Y,Jin ML,et al.A novel multifunctional C-23 oxidase,CYP714E19,is involved in asiaticoside biosynthesis[J].Plant Cell Physiol,2018,59(6):1200-1213.
[22] Carelli M,Biazzi E,Panara F,et al.Medicago truncatula CYP716A12 is a multifunctional oxidase involved in the biosynthesis of hemolytic saponins[J].Plant Cell,2011,23(8):3070-3081.
[23] Han J,Kim M,Ban Y,et al.The involvement of beta-amyrin 28-oxidase (CYP716A52v2) in oleanane-type ginsenoside biosynthesis in Panax ginseng[J].Plant Cell Physiol,2013,54(12):2034-2046.