布渣叶糖基转移酶MpUGT1基因cDNA克隆及生物信息学和表达分析
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摘要
UGT催化的糖基转移反应对合成黄酮苷类至关重要。为探究影响布渣叶黄酮苷生物合成的糖基转移酶,本研究参考布渣叶转录组数据,设计特异性引物,采用RT-PCR法对布渣叶中的UDP-葡萄糖基转移酶(UDP-glucosyltransferase, UGT)基因MpUGT1的编码区进行克隆,并通过在线服务器分析该基因的生物信息学特性;利用实时荧光定量PCR方法检测了MpUGT1基因在布渣叶的芽、叶、枝、果、花不同部位的表达情况。结果表明,本研究克隆得到的布渣叶MpUGT1基因,其cDNA全长为1 397 bp,ORF 1 377 bp,编码458个氨基酸,GenBank登陆号为KY652922。生物信息学预测分析该基因编码蛋白质分子式为C2324H3643N603O671S18,相对分子质量为51 kD,理论等电点(PI)为5.99,不稳定系数为43.81,亲水性系数为-0.105。该蛋白没有跨膜结构域,含有UDP-葡萄糖醛酸/UDP-葡萄糖基转移酶家族保守结构域,不含信号肽,定位于叶绿体。RT-qPCR组织特异性表达分析结果表明MpUGT1在芽、叶、枝、果、花中均有表达,且叶的表达量最高。系统进化树分析显示Mp UGT1与同科植物黄麻、长蒴黄麻等同源UGT的相似度最高,均达到了约80%;上述结果为进一步研究该基因在布渣叶黄酮苷生物合成中的作用奠定了基础。
UGT catalyzed glycosylation reactions are essential for the synthesis of flavonoid glycosides. In order to explore an UDP-glucosyltransferase(UGT) that affects the biosynthesis of flavonoid glycosides in M. paniculata,this study designed specific primers based on the transcriptome data of M. paniculata, and cloned the coding region of UDP-glucosyltransferase gene MpUGT1 in M. paniculata by RT-PCR. The bioinformatics characteristics of MpUGT1 gene was analyzed through online server; and the expression of MpUGT1 gene in M. paniculata buds,leaves, twigs, fruits and flowers was detected by RT-qPCR. The results showed that the cloned MpUGT1 gene in this study was 1 397 bp with an open reading frame of 1 377 bp encoding 458 amino acids. The GenBank accession number was KY652 922. Bioinformatics analysis indicated that the gene encoding protein molecular formula was C2324 H3643 N603 O671 S18 and the relative molecular weight was 51.3 kD, its theoretical isoelectric point(PI)was 5.99 and the instability coefficient was 43.81, and the hydrophilicity coefficient was-0.105. MpUGT1 protein located in chloroplast without transmembrane domain and signal peptide, and contained the UDP-glucuronosyl/UDPglucosyltransferase family conserved domain. Tissue-specific expression analysis of RT-qPCR indicated that MpUGT1 was expressed in buds, leaves, twigs, fruits and flowers, and the highest expression level of MpUGT1 was in leaves. Phylogenetic tree analysis showed that MpUGT1 might have the high homology with homologous plants such as C. capsularis and C. olitoriu, which are both up to 80%. The result would be a foundation for the further study of the function of MpUGT1 gene in the biosynthesis of flavone glycosides in M. paniculata.
UGT catalyzed glycosylation reactions are essential for the synthesis of flavonoid glycosides. In order to explore an UDP-glucosyltransferase(UGT) that affects the biosynthesis of flavonoid glycosides in M. paniculata,this study designed specific primers based on the transcriptome data of M. paniculata, and cloned the coding region of UDP-glucosyltransferase gene MpUGT1 in M. paniculata by RT-PCR. The bioinformatics characteristics of MpUGT1 gene was analyzed through online server; and the expression of MpUGT1 gene in M. paniculata buds,leaves, twigs, fruits and flowers was detected by RT-qPCR. The results showed that the cloned MpUGT1 gene in this study was 1 397 bp with an open reading frame of 1 377 bp encoding 458 amino acids. The GenBank accession number was KY652 922. Bioinformatics analysis indicated that the gene encoding protein molecular formula was C2324 H3643 N603 O671 S18 and the relative molecular weight was 51.3 kD, its theoretical isoelectric point(PI)was 5.99 and the instability coefficient was 43.81, and the hydrophilicity coefficient was-0.105. MpUGT1 protein located in chloroplast without transmembrane domain and signal peptide, and contained the UDP-glucuronosyl/UDPglucosyltransferase family conserved domain. Tissue-specific expression analysis of RT-qPCR indicated that MpUGT1 was expressed in buds, leaves, twigs, fruits and flowers, and the highest expression level of MpUGT1 was in leaves. Phylogenetic tree analysis showed that MpUGT1 might have the high homology with homologous plants such as C. capsularis and C. olitoriu, which are both up to 80%. The result would be a foundation for the further study of the function of MpUGT1 gene in the biosynthesis of flavone glycosides in M. paniculata.
引文
Chen Y.F.,Yang C.Y.,Li K.P.,Zeng Y.,Wang Y.F.,and Chen M.Y.,2013,Protection of total flavones from Microcos paniculata on acute myocardial ischemia in rats and its mechanism,Zhongcaoyao(Chinese Traditional and Herbal Drugs)35(8):1003-1007(陈艳芬,杨超燕,李坤平,曾颖,王颖芳陈美燕,2013,布渣叶总黄酮对大鼠急性心肌缺血的保护作用及其机制,中草药,35(8):1003-1007)
Ferreyra M.L.F.,Rius S.P.,and Casati P.,2012,Flavonoids:biosynthesis,biological functions,and biotechnological applications,Frontiers in Plant Science,3(222):222
García-Lafuente A.,Guillamón E.,Villares A.,Rostagno M.A.and Martínez J.A.,2009,Flavonoids as anti-inflammatory agents:implications in cancer and cardiovascular disease,Inflammation Research,58(9):537-552
Hall D.,Yuan X.X.,Murata J.,and De Luca V.,2012,Molecular cloning and biochemical characterization of the UDP-glucose:Flavonoid 3-O-glucosyltransferase from Concord grape(Vitis labrusca),Phytochemistry,74(3):90-99
Li K.P.,Pan T.L.,Gao C.K.,and Li W.M.,2009,Study on the enrichment and purification of total flavonoids in Microcos paniculata by macroporous adsorption resin,Zongyaoc a(Journal of Chinese Medicinal Materials),32(4):601-604(李坤平,潘天玲,高崇凯,李卫民,2009,大孔吸附树脂富集纯化布渣叶总黄酮的研究,中药材,32(4):601-604)
Lu C.,Zhao S.,Wei G.,Zhao H.,and Qu Q.,2016,Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius,PlantPhysiology&Biochemistry,111:67-76
Sui X.,Gao X.,Ao M.,Wang Q.,Yang D.,Wang M.,Fu Y.,and Wang L.,2011,cDNA cloning and characterization of UDP-glucose:anthocyanidin 3-O-glucosyltransferase in Freesia hybrida,Plant Cell Rep.,30(7):1209-1218
Sun D.M.,and Wang M.X.,2015,Advances on chemical constituents and pharmacological effects of Microcos paniculata Shijie Zhongyiyao(World Chinese Medicine),10(1):143-147(孙冬梅,汪梦霞,2015,布渣叶化学成分和药理作用研究进展,世界中医药,10(1):143-147)
Tang Y.P.,Yuan H.,and Qin J.B.,2012,Identification and characterization of a flavonoid-3-O-glucosyltransferas gene from Saussurea involucrata,Shengwu Gongcheng Xuebao(Chinese Journal of Biotechnology),28(6):705-714(唐亚萍,原慧,覃建兵,2012,天山雪莲UDP葡萄糖-类黄酮-3-O-葡萄糖基转移酶基因的克隆及功能分析,生物工程学报,28(6):705-714)
Tu Y.S.,Zhu Y.,Sun D.M.,Wang L.L.,and Xu W.J.,2014,Solid dispersion of microtis folium flavonoids prepared by hot-melt extrusion and its characterization research,Zhongyaocai(Journal of Chinese Medicinal Materials),37(3):508-512(涂瑶生,朱颖,孙冬梅,王洛临,徐文杰,2014,热熔挤出技术制备布渣叶总黄酮固体分散体及其表征研究,中药材,37(3):508-512)
Veljanovski V.,and Constabel C.P.,2013,Molecular cloning and biochemical characterization of two UDP-glycosyltransferases from poplar,Phytochemistry,91(4):148-157
Wu Y.Y.,Zhao X.L.,Chen P.,and Zhang S.P.,2017,Cloning ofβ-amyrin synthase gene from Panax japonicus var.major and its bioinformatics analysis,Shengwu Jishu Tongbao(Biotechnology Bulletin),33(2):109-117(吴亚运,赵小龙,陈平,张绍鹏,2017,珠子参β-香树素合成酶基因的克隆和生物信息学分析,生物技术通报,33(2):109-117)
Xing A.J.,Ma X.J.,Mo C.M.,Pan L.M.,Wei P.X.,Tang C.F.,and Tang Q.,2013,Cloning and prokaryotic expression of UDP-glycosyltransferase in Siraitia grosvenorii,Yuanyi Xuebao(Acta Horticulturae Sinica),40(6):1195-1204(邢爱佳,马小军,莫长明,潘丽梅,韦鹏霄,唐春风,唐其,2013,罗汉果葡萄糖基转移酶基因的克隆及原核表达,园艺学报,40(6):1195-1204)
Xu D.H.,and Tan C.Y.,2016,Gene cloning and prokaryotic expression of glycosyltransferase from Leonurus heterophyllus Sweet,Zhiwu Kexue Xuebao(Plant Sclence Journal),34(3):397-405(徐德宏,谭朝阳,2016,益母草糖基转移酶基因的克隆及原核表达分析,植物科学学报,34(3):397-405)
Xu J.S.,Wei J.H.,Tao Y.W.,Sun J.,and Sui C.,2013,Cloning,sequence analysis,and prokaryotic expression vector construction of glycosyltransferase gene BcUGT8 from Bupleurum chinense,Zhongcaoyao(Chinese Traditional and Herbal Drugs),35(17):2453-2459(徐洁森,魏建和,陶韵文,孙晶,隋春,2013,北柴胡糖基转移酶基因BcUGT8的克隆,序列分析及其原核表达载体构建,中草药,35(17):2453-2459)
Yonekura-Sakakibara K.,Nakabayashi R.,Sugawara S.,Tohge T.,Ito T.,Koyanagi M.,Kitajima M.,Takayama H.,and Sait o K.,2014,A flavonoid 3-O-glucoside:2"-O-glucosyltransferase responsible for terminal modification of pollen-specific flavonols in Arabidopsis thaliana,Plant Journal for Cell&Molecular Biology,79(5):769-782
Ferreyra M.L.F.,Rius S.P.,and Casati P.,2012,Flavonoids:biosynthesis,biological functions,and biotechnological applications,Frontiers in Plant Science,3(222):222
García-Lafuente A.,Guillamón E.,Villares A.,Rostagno M.A.and Martínez J.A.,2009,Flavonoids as anti-inflammatory agents:implications in cancer and cardiovascular disease,Inflammation Research,58(9):537-552
Hall D.,Yuan X.X.,Murata J.,and De Luca V.,2012,Molecular cloning and biochemical characterization of the UDP-glucose:Flavonoid 3-O-glucosyltransferase from Concord grape(Vitis labrusca),Phytochemistry,74(3):90-99
Li K.P.,Pan T.L.,Gao C.K.,and Li W.M.,2009,Study on the enrichment and purification of total flavonoids in Microcos paniculata by macroporous adsorption resin,Zongyaoc a(Journal of Chinese Medicinal Materials),32(4):601-604(李坤平,潘天玲,高崇凯,李卫民,2009,大孔吸附树脂富集纯化布渣叶总黄酮的研究,中药材,32(4):601-604)
Lu C.,Zhao S.,Wei G.,Zhao H.,and Qu Q.,2016,Functional regulation of ginsenoside biosynthesis by RNA interferences of a UDP-glycosyltransferase gene in Panax ginseng and Panax quinquefolius,PlantPhysiology&Biochemistry,111:67-76
Sui X.,Gao X.,Ao M.,Wang Q.,Yang D.,Wang M.,Fu Y.,and Wang L.,2011,cDNA cloning and characterization of UDP-glucose:anthocyanidin 3-O-glucosyltransferase in Freesia hybrida,Plant Cell Rep.,30(7):1209-1218
Sun D.M.,and Wang M.X.,2015,Advances on chemical constituents and pharmacological effects of Microcos paniculata Shijie Zhongyiyao(World Chinese Medicine),10(1):143-147(孙冬梅,汪梦霞,2015,布渣叶化学成分和药理作用研究进展,世界中医药,10(1):143-147)
Tang Y.P.,Yuan H.,and Qin J.B.,2012,Identification and characterization of a flavonoid-3-O-glucosyltransferas gene from Saussurea involucrata,Shengwu Gongcheng Xuebao(Chinese Journal of Biotechnology),28(6):705-714(唐亚萍,原慧,覃建兵,2012,天山雪莲UDP葡萄糖-类黄酮-3-O-葡萄糖基转移酶基因的克隆及功能分析,生物工程学报,28(6):705-714)
Tu Y.S.,Zhu Y.,Sun D.M.,Wang L.L.,and Xu W.J.,2014,Solid dispersion of microtis folium flavonoids prepared by hot-melt extrusion and its characterization research,Zhongyaocai(Journal of Chinese Medicinal Materials),37(3):508-512(涂瑶生,朱颖,孙冬梅,王洛临,徐文杰,2014,热熔挤出技术制备布渣叶总黄酮固体分散体及其表征研究,中药材,37(3):508-512)
Veljanovski V.,and Constabel C.P.,2013,Molecular cloning and biochemical characterization of two UDP-glycosyltransferases from poplar,Phytochemistry,91(4):148-157
Wu Y.Y.,Zhao X.L.,Chen P.,and Zhang S.P.,2017,Cloning ofβ-amyrin synthase gene from Panax japonicus var.major and its bioinformatics analysis,Shengwu Jishu Tongbao(Biotechnology Bulletin),33(2):109-117(吴亚运,赵小龙,陈平,张绍鹏,2017,珠子参β-香树素合成酶基因的克隆和生物信息学分析,生物技术通报,33(2):109-117)
Xing A.J.,Ma X.J.,Mo C.M.,Pan L.M.,Wei P.X.,Tang C.F.,and Tang Q.,2013,Cloning and prokaryotic expression of UDP-glycosyltransferase in Siraitia grosvenorii,Yuanyi Xuebao(Acta Horticulturae Sinica),40(6):1195-1204(邢爱佳,马小军,莫长明,潘丽梅,韦鹏霄,唐春风,唐其,2013,罗汉果葡萄糖基转移酶基因的克隆及原核表达,园艺学报,40(6):1195-1204)
Xu D.H.,and Tan C.Y.,2016,Gene cloning and prokaryotic expression of glycosyltransferase from Leonurus heterophyllus Sweet,Zhiwu Kexue Xuebao(Plant Sclence Journal),34(3):397-405(徐德宏,谭朝阳,2016,益母草糖基转移酶基因的克隆及原核表达分析,植物科学学报,34(3):397-405)
Xu J.S.,Wei J.H.,Tao Y.W.,Sun J.,and Sui C.,2013,Cloning,sequence analysis,and prokaryotic expression vector construction of glycosyltransferase gene BcUGT8 from Bupleurum chinense,Zhongcaoyao(Chinese Traditional and Herbal Drugs),35(17):2453-2459(徐洁森,魏建和,陶韵文,孙晶,隋春,2013,北柴胡糖基转移酶基因BcUGT8的克隆,序列分析及其原核表达载体构建,中草药,35(17):2453-2459)
Yonekura-Sakakibara K.,Nakabayashi R.,Sugawara S.,Tohge T.,Ito T.,Koyanagi M.,Kitajima M.,Takayama H.,and Sait o K.,2014,A flavonoid 3-O-glucoside:2"-O-glucosyltransferase responsible for terminal modification of pollen-specific flavonols in Arabidopsis thaliana,Plant Journal for Cell&Molecular Biology,79(5):769-782
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