马尾松PmCOMT基因的克隆及实时定量表达分析
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摘要
在木质素生物合成里,咖啡酸-O-甲基转移酶(caffeic acid-O-methyltransferase, COMT)起重要作用。本论文以马尾松幼苗作为材料,并通过PCR技术以及RACE技术从马尾松材料中克隆出Pm COMT基因的cDNA全长。该cDNA全长为1660bp,其中预测完整ORF全长为1125bp,可编码374个氨基酸的蛋白。蛋白相对分子质量为41.81 kD;等电点(pI)为5.03,总平均亲水性为-0.311。通过生物信息学的方法对其进行分析表明,Pm COMT基因具有典型的甲基转移酶的保守结构域。通过实时荧光定量表达分析表明,COMT基因在马尾松的不同组织均有表达,其中在新梢中的表达量最高,树皮中表达量最低。
Caffeic acid-O-methyltransferase(COMT) plays an important role in the lignin biosynthesis. With the seedlings of Pinus massoniana as materials, we used PCR and RACE to clone the full-length cDNA of Pm COMT gene from Pinus massoniana. The full length of this cDNA was 1 660 bp, the predicted full length of intact open reading frame was 1 125 bp and 374 amino acids could be encoded. The relative molecular mass of the protein was41.81 kD, the isoelectric point(pI) was 5.03, and the total average hydrophilicity was-0.311. Analysis by bioinformatics way showed that the Pm COMT gene had a conserved domain of typical methyltransferase. Real-time fluorescence quantitative expression analysis showed that Pm COMT gene was expressed in different tissues of Pinus massoniana, and its expression was the highest in new shoots and the lowest in bark.
Caffeic acid-O-methyltransferase(COMT) plays an important role in the lignin biosynthesis. With the seedlings of Pinus massoniana as materials, we used PCR and RACE to clone the full-length cDNA of Pm COMT gene from Pinus massoniana. The full length of this cDNA was 1 660 bp, the predicted full length of intact open reading frame was 1 125 bp and 374 amino acids could be encoded. The relative molecular mass of the protein was41.81 kD, the isoelectric point(pI) was 5.03, and the total average hydrophilicity was-0.311. Analysis by bioinformatics way showed that the Pm COMT gene had a conserved domain of typical methyltransferase. Real-time fluorescence quantitative expression analysis showed that Pm COMT gene was expressed in different tissues of Pinus massoniana, and its expression was the highest in new shoots and the lowest in bark.
引文
Atanassova R.,Favet N.,and Martz F.,1995,Altered lignin composition in transgenic tobacco expressing O-methyltransferase sequences in sense and antisense orientation,Plant J.8(4):465-477
Chen B.H.,2009,Cloning and sequence analysis of cinnamoyl-C-oA reductase gene(CCR)of Pinus massoniana,Scientia Silvae Sinicae,45(12):46-53
Dwivedi U.N.,Campbell W.H.,and Yu J.,1995,Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus Plant Molecular Biology,26(1):61-71
Han X.,2012,Gene cloning of Pinus massoniana 4CL construc-tion of its RNAi expression vector,Thesis for M.S.,Centra South University of Forestry and Technology,Supervisor Zhang D.Q.,pp.1-11(韩欣,2012,马尾松木质素合成途径中4CL基因克隆及RNA干扰载体构建研究,硕士学位论文中南科技林业大学,导师:张党权,pp.1-11)
Jing K.W.,Takuya A.,and John R.,2011,Independent recruitmen of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii,The Plant Cell,23(7):2708-2724
Li B.,Ni Z.Y.,Wang J.,and Lv M.,2010,Advances on key enzyme gene(COMT)involved in lignin biosynthesis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(1):117-124(李波倪志勇,王娟,2010,木质素生物合成关键酶咖啡酸-O-甲基转移酶基因(COMT)的研究进展,分子植物育种,8(1):117-124)
Luo J.,Wang Y.Q.,and Wen S.C.,2016,Gene cloning and sequence analysis of caffeic acid O-methyltransferase in Angelica sinensis,Zhongcaoyao(Chinese Traditional and Herbal Drugs)47(7):1180-1186(雒军,王引权,温随超,2016,当归咖啡-O-甲基转移酶基因克隆和序列分析,中草药,47(7):1180-1186)
Ma Q.H.,and Xu Y.,2008,Characterization of a caffeic acid 3-O-methyltransferase from wheat and its function in lignin biosynthesis,Biochimie,90(3):515-524
Meyermans H.,Morreel K.,and Lapierre C.,2000,Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme a O-methyltransferase,an enzyme involved in lignin biosynthesis The Journal of Biological Chemistry,275(47):36899-36909
Qiao M.X.,Lin X.F.,and Zhang W.B.,2016,Isolation and char acterization of caffeic acid O-methyltransferase gene from Larix gmelinii,Fenzi Zhiwu Yuzhong(Molecular Plant Bree ding),14(7):1684-1690(乔明星,林晓飞,张文波,2016,兴安落叶松咖啡酸-O-甲基转移酶基因的克隆及特性分析分子植物育种,47(4):1684-1690)
Qiu W.H.,and Chen H.Z.,2006,Structure,function and highe value application of lignin,Qianweisu Kexue Yu Jishu(Journal of Cellulose Science and Technology),14(1):52-59(邱卫华,陈洪章,2006,木质素的结构、功能及高值化利用,纤维素科学与技术,14(1):52-59)
Wang M.,Cao F.X.,Wang S.L.,and Long J.X.,2010,Molecula cloning and sequence analysis of phenylalanine ammoni alyase cDNA from Pinus massoniana,Zhongnanlinye Keji daxue Xuebao(Journal of Central South University of Forestry&Technology),30(1):79-83(王猛,曹福祥,王仕利,龙绛雪,2010,马尾松苯丙氨酸解氨酶基因的克隆与序列分析,中南林业科技大学学报,30(1):79-83)
Wei J.H.,and Song Y.R.,2001,Advances in study of lignin biosynthsis and manipulation,Acta Botanica Sinica,43(8):771779
Wei J.H.,and Song Y.R.,2001,Recent advances in study o lignin biosynthesis and manipulation,Zhiwu Xuebao(Act Botanica Sinica),43(8):771-779(魏建华,宋艳茹,2001,木质素生物合成途径及调控的研究进展,植物学报,43(8)771-779)
Zein I.,Wenzel G.,Andersen J.R.,and Lubberstedt T.,2007,Low level of linkage disequilibrium at the COMT(caffeicacid Om ethyltransferase)locus in European maize,Zea mays L.Genetic Resources and Crop Evolution,54(1):139-148
Zhang F.K.,2014,Cloning and analyzing of CAD and CCoAOMTgenes from Pinus massoniana,Thesis for M.S.,Nanjing Forestry Univercity,Supervisor:Ji K.S.,pp.10-31(张逢凯,2014马尾松CAD和CCo AOMT基因的克隆与表达分析,硕士学位论文,南京林业大学,导师:季孔庶,pp.10-31)
Zhou J.M.,Yong W.S.,and Ragai K.,2009,Biochemical charac terization of a putative wheat caffeic acid O-methyltrans ferase,Plant Physiology and Biochemistry,47(4):322-326
Zhu J.Q.,Xiang M.,and Li Y.Y.,2016,Molecular cloning and sequence analysis of caffeic acid O-methyltransferase in Li gusticum chuanxiong Hort,Tianranchanwu Yanjiu Yu Kaif(Natural Product Research and Development),28(10):15051509(朱建全,向缅,李洋洋,2016,川芎咖啡酸-O-甲基转移酶基因的克隆及序列分析,天然产物研究与开发,28(10):1505-1509)
Chen B.H.,2009,Cloning and sequence analysis of cinnamoyl-C-oA reductase gene(CCR)of Pinus massoniana,Scientia Silvae Sinicae,45(12):46-53
Dwivedi U.N.,Campbell W.H.,and Yu J.,1995,Modification of lignin biosynthesis in transgenic Nicotiana through expression of an antisense O-methyltransferase gene from Populus Plant Molecular Biology,26(1):61-71
Han X.,2012,Gene cloning of Pinus massoniana 4CL construc-tion of its RNAi expression vector,Thesis for M.S.,Centra South University of Forestry and Technology,Supervisor Zhang D.Q.,pp.1-11(韩欣,2012,马尾松木质素合成途径中4CL基因克隆及RNA干扰载体构建研究,硕士学位论文中南科技林业大学,导师:张党权,pp.1-11)
Jing K.W.,Takuya A.,and John R.,2011,Independent recruitmen of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii,The Plant Cell,23(7):2708-2724
Li B.,Ni Z.Y.,Wang J.,and Lv M.,2010,Advances on key enzyme gene(COMT)involved in lignin biosynthesis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),8(1):117-124(李波倪志勇,王娟,2010,木质素生物合成关键酶咖啡酸-O-甲基转移酶基因(COMT)的研究进展,分子植物育种,8(1):117-124)
Luo J.,Wang Y.Q.,and Wen S.C.,2016,Gene cloning and sequence analysis of caffeic acid O-methyltransferase in Angelica sinensis,Zhongcaoyao(Chinese Traditional and Herbal Drugs)47(7):1180-1186(雒军,王引权,温随超,2016,当归咖啡-O-甲基转移酶基因克隆和序列分析,中草药,47(7):1180-1186)
Ma Q.H.,and Xu Y.,2008,Characterization of a caffeic acid 3-O-methyltransferase from wheat and its function in lignin biosynthesis,Biochimie,90(3):515-524
Meyermans H.,Morreel K.,and Lapierre C.,2000,Modifications in lignin and accumulation of phenolic glucosides in poplar xylem upon down-regulation of caffeoyl-coenzyme a O-methyltransferase,an enzyme involved in lignin biosynthesis The Journal of Biological Chemistry,275(47):36899-36909
Qiao M.X.,Lin X.F.,and Zhang W.B.,2016,Isolation and char acterization of caffeic acid O-methyltransferase gene from Larix gmelinii,Fenzi Zhiwu Yuzhong(Molecular Plant Bree ding),14(7):1684-1690(乔明星,林晓飞,张文波,2016,兴安落叶松咖啡酸-O-甲基转移酶基因的克隆及特性分析分子植物育种,47(4):1684-1690)
Qiu W.H.,and Chen H.Z.,2006,Structure,function and highe value application of lignin,Qianweisu Kexue Yu Jishu(Journal of Cellulose Science and Technology),14(1):52-59(邱卫华,陈洪章,2006,木质素的结构、功能及高值化利用,纤维素科学与技术,14(1):52-59)
Wang M.,Cao F.X.,Wang S.L.,and Long J.X.,2010,Molecula cloning and sequence analysis of phenylalanine ammoni alyase cDNA from Pinus massoniana,Zhongnanlinye Keji daxue Xuebao(Journal of Central South University of Forestry&Technology),30(1):79-83(王猛,曹福祥,王仕利,龙绛雪,2010,马尾松苯丙氨酸解氨酶基因的克隆与序列分析,中南林业科技大学学报,30(1):79-83)
Wei J.H.,and Song Y.R.,2001,Advances in study of lignin biosynthsis and manipulation,Acta Botanica Sinica,43(8):771779
Wei J.H.,and Song Y.R.,2001,Recent advances in study o lignin biosynthesis and manipulation,Zhiwu Xuebao(Act Botanica Sinica),43(8):771-779(魏建华,宋艳茹,2001,木质素生物合成途径及调控的研究进展,植物学报,43(8)771-779)
Zein I.,Wenzel G.,Andersen J.R.,and Lubberstedt T.,2007,Low level of linkage disequilibrium at the COMT(caffeicacid Om ethyltransferase)locus in European maize,Zea mays L.Genetic Resources and Crop Evolution,54(1):139-148
Zhang F.K.,2014,Cloning and analyzing of CAD and CCoAOMTgenes from Pinus massoniana,Thesis for M.S.,Nanjing Forestry Univercity,Supervisor:Ji K.S.,pp.10-31(张逢凯,2014马尾松CAD和CCo AOMT基因的克隆与表达分析,硕士学位论文,南京林业大学,导师:季孔庶,pp.10-31)
Zhou J.M.,Yong W.S.,and Ragai K.,2009,Biochemical charac terization of a putative wheat caffeic acid O-methyltrans ferase,Plant Physiology and Biochemistry,47(4):322-326
Zhu J.Q.,Xiang M.,and Li Y.Y.,2016,Molecular cloning and sequence analysis of caffeic acid O-methyltransferase in Li gusticum chuanxiong Hort,Tianranchanwu Yanjiu Yu Kaif(Natural Product Research and Development),28(10):15051509(朱建全,向缅,李洋洋,2016,川芎咖啡酸-O-甲基转移酶基因的克隆及序列分析,天然产物研究与开发,28(10):1505-1509)