光皮桦BlCCoAOMT基因的克隆、表达及单核苷酸变异分析
|
摘要
咖啡酰辅酶A-O-甲基转移酶(CCoAOMT)是植物木质素合成过程中的一个关键酶,对木质素组成和结构有重要的作用。为探究BlCCoAOMT基因在木质素合成中的作用,本研究从光皮桦中克隆了该基因,并分析其基因结构和表达模式,以及在73个基因型中的单核苷酸多态性(SNP)。结果表明,BlCCoAOMT基因cDNA全长1 114 bp,含有一个744 bp的完整ORF,编码一个由247个氨基酸残基组成的蛋白;该编码蛋白包含高等植物CCoAOMT所有的8个典型保守基序。BlCCoAOMT在木质化茎段中优势表达,且随着木质化程度提高而逐渐增强,表明BlCCoAOMT可能参与光皮桦木质素的生物合成;在拉弯处理的6 h到7 d间,该基因在应拉区发育木质部中的表达显著下调,与应拉木木质素含量的下降相符。共检测到BlCCoAOMT基因有119个SNP位点,平均每14 bp就有1个SNP位点,其中在外显子区域存在26个SNP位点,表明不同基因型中存在丰富SNP变异;在不同群体间BlCCoAOMT基因的分化程度无显著差异,且非同义突变和同义突变的位点的比值均小于1,表明在演化进程中主要受到了纯化选择压力。本研究结果为深入解析光皮桦木质素合成分子机制及后续分子标记辅助育种提供了理论依据。
Caffeoyl coenzyme A 3-O-methyltransferase( CCoAOMT) is a key enzyme involved in lignin biosynthesis,has an important influence on the composition and structure of lignin. To investigate its function in lignin biosynthesis,a BlCCoAOMT gene was cloned from Betula luminifera,and its sequence characteristics and expression patterns were analyzed in this study. The genomic sequences of BlCCoAOMT from 73 individuals were analyzed for single nucleotide polymorphism( SNP) detection. The results showed that the BlCCoAOMT cDNA was 1114 bp in length with an open reading frame( ORF) of 744 bp. The encoding protein was composed of 247 amino acid residues,which contained all eight conserved motifs of plant CCoAOMTs. The expression levels of BlCCoAOMT were relatively high in lignified stem,and increased with the lignification progressed,suggesting that it might participate in lignin biosynthesis of B. luminifera.The gene expression in TW was significantly down-regulated with the process of bending from 6 hours to 7 days which was consistent with the decrease of the lignin content in TW. In total,119 SNPs were detected,one SNP among which26 SNPs located in exons of BlCCoAOMT. The frequency of SNP was one SNP per 14 bp on average,indicating that this gene had abundant SNPs among different genotypes. Genetic divergence of BlCCoAOMT was insignificantly different among the populations,and the ratio of nonsynonymous to synonymous mutations was less than 1,suggesting that BlCCoAOMT was mainly subjected to purification selection in the evolution process. The present study laid a foundation for in-depth dissection of lignin biosynthesis mechanism and provided theory basis for further molecular marker assisted breeding in B. luminifera.
Caffeoyl coenzyme A 3-O-methyltransferase( CCoAOMT) is a key enzyme involved in lignin biosynthesis,has an important influence on the composition and structure of lignin. To investigate its function in lignin biosynthesis,a BlCCoAOMT gene was cloned from Betula luminifera,and its sequence characteristics and expression patterns were analyzed in this study. The genomic sequences of BlCCoAOMT from 73 individuals were analyzed for single nucleotide polymorphism( SNP) detection. The results showed that the BlCCoAOMT cDNA was 1114 bp in length with an open reading frame( ORF) of 744 bp. The encoding protein was composed of 247 amino acid residues,which contained all eight conserved motifs of plant CCoAOMTs. The expression levels of BlCCoAOMT were relatively high in lignified stem,and increased with the lignification progressed,suggesting that it might participate in lignin biosynthesis of B. luminifera.The gene expression in TW was significantly down-regulated with the process of bending from 6 hours to 7 days which was consistent with the decrease of the lignin content in TW. In total,119 SNPs were detected,one SNP among which26 SNPs located in exons of BlCCoAOMT. The frequency of SNP was one SNP per 14 bp on average,indicating that this gene had abundant SNPs among different genotypes. Genetic divergence of BlCCoAOMT was insignificantly different among the populations,and the ratio of nonsynonymous to synonymous mutations was less than 1,suggesting that BlCCoAOMT was mainly subjected to purification selection in the evolution process. The present study laid a foundation for in-depth dissection of lignin biosynthesis mechanism and provided theory basis for further molecular marker assisted breeding in B. luminifera.
引文
[1] Li X F,Li X,Jia B,Liu L,Ye Z F,Heng W,Zhu L W. Analysisof enzyme activity and lignin content and expression of CCoAOMTgene in the pericarp of ‘Dangshan Suli’and its russet mutant[J].ACTA Horticulturae Sinica,2012,39(5):828-835
[2]李潞滨,刘蕾,何聪芬,董银卯,彭镇华.木质素生物合成关键酶基因的研究进展[J].分子植物育种,2007,5(S1):45-51
[3] Hao Z,Mohnen D. A review of xylan and lignin biosynthesis:foundation for studying Arabidopsis irregular xylem mutants withpleiotropic phenotypes[J]. Critical Reviews in Biochemistry&Molecular Biology,2014,49(3):212-241
[4] Wang H,Yu Y,Fu C X,Zhou G K,Gao H H. Progress of a keyenzyme caffeoyl-CoA 3-O-methyltransferase in lignin biosynthesis[J]. Genomics&Applied Biology,2014,33(2):458-466
[5] Hu D,Liu X B,She H Z,Hu D,Liu X B,She H Z,Gao Z,RuanR,Wu D Q,Yi Z L. The lignin synthesis related genes and lodgingresistance of Fagopyrum esculentum[J]. Biologia Plantarum,2017,61(1):138-146
[6] Xu R X,Zhao Y,Gao S,Zhang Y Y,Li D D,Lou H X,Cheng AX. Functional characterization of a plastidal cation-dependent O-methyltransferase from the liverwort Plagiochasma appendiculatum[J]. Phytochemistry,2015,8(2):33-41
[7] Do C T,Pollet B,Thévenin J,Sibout R,Denoue D,Barrière Y,Catherine L, Jouanin L. Both caffeoyl coenzyme a 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 areinvolved in redundant functions for lignin,flavonoids and sinapoylmalate biosynthesis in Arabidopsis[J]. Planta,2007,226(5):1117-1129
[8] Li X Y,Chen W J,Zhao Y,Xiang Y,Jiang H Y,Zhu S W,ChengB J. Downregulation of caffeoyl-CoA O-methyltransferase(CCoAOMT)by RNA interference leads to reduced ligninproduction in maize straw[J]. Genetics&Molecular Biology,2013,36(4):540-546
[9] Zhong R,Morrison W H,Himmelsbach D S,Poole F L,Ye Z H.Essential role of caffeoyl coenzyme a O-methyltransferase in ligninbiosynthesis in woody poplar plants[J]. Plant Physiology,2000,124(2):563-578
[10] Liu S J,Huang Y H,He C J,Fang C,Zhang Y W. Cloning,bioinformatics and transcriptional analysis of caffeoyl coenzyme a 3-O-methyltransferase in switchgrass under abiotic stress[J]. Journalof Integrative Agriculture,2016,15(3):636-649
[11] FornaléS, Rencoret J, García-calvo L, Encina A, Rigau J,Gutiérrez A,Río J C D,Caparros-Ruiz D. Changes in cell wallpolymers and degradability in maize mutants lacking 3'-and 5'-O-methyltransferases involved in lignin biosynthesis[J]. Plant&CellPhysiology,2016,58(2):240-255
[12]杨业斌.光皮桦的经济性状及栽培技术[J].湖南林业科技,1990(4):20-21
[13]黄华宏.基于转录组测序的光皮桦应拉木形成分子机制研究[D].杭州:浙江大学,2012:21-28
[14] Huang H H,Jiang C,Tong Z K,Cheng L J,Zhu M Y,Lin E P.Eight distinct cellulose synthase catalytic subunit genes from Betulaluminifera are associated with primary and secondary cell wallbiosynthesis[J]. Cellulose,2014,21(4):2183-2198
[15]江成.光皮桦BlOFPs基因的克隆及其功能研究[D].杭州:浙江农林大学,2014:21-22,50
[16]赵宏亮,陈凯,张强,徐正进,黎志康.基于连锁不平衡水稻源库相关性状的关联分析[J].核农学报,2015,29(4):674-684
[17]巩琛锐,王璐,杜庆章,张德强.毛白杨S-腺苷高半胱氨酸水解酶基因PtSAHHA的克隆、表达及SNP关联分析[J].林业科学,2013,49(11):67-76
[18] Colasuonno P,Gadaleta A,Giancaspro A,Nigro D,Giove S,Incerti O, Mangini G, Signorile A, Simeone R, Blanco A.Development of a high-density SNP-based linkage map anddetection of yellow pigment content QTLs in durum wheat[J].Molecular Breeding,2014,34(4):1563-1578
[19]何辉,楼雄珍,林二培,俞友明,童再康,黄华宏.光皮桦应拉木的显微特征及其形成早期内源激素分布[J].林业科学,2016,52(10):38-44
[20]张俊红,黄华宏,童再康,程龙军,梁跃龙,陈奕良.光皮桦6个南方天然群体的遗传多样性[J].生物多样性,2010,18(3):233-240
[21] Kumar S,Stecher G,Tamura K. MEGA7:molecular evolutionarygenetics analysis version 7. 0 for bigger datasets[J]. MolecularBiology and Evolution,2016,33(7):1870-1874
[22] Librado P, Rozas J. DnaSP v5:a software for comprehensiveanalysis of DNA polymorphism data[J]. Bioinformatics,2009,25(11):1451-1452
[23] Joshi C P,ChiangⅤL. Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases[J]. PlantMolecular Biology,1998,37(4):663
[24] Mizrachi E,MaloneyⅤJ,Silberbauer J,Hefer C A,Berger D K,Mansfield S D, Myburg A A. Investigating the molecularunderpinnings underlying morphology and changes in carbonpartitioning during tension wood formation in Eucalyptus[J]. NewPhytologist,2015,206(4):1351-1363
[25] Chang S S,Salmén L,Olsson A M,Clair B. Deposition andorganization of cell wall polymers during maturation of poplar tensionwood by FTIR microspectroscopy[J]. Planta,2014,239(1):243-254
[26] Esteras C,Formisano G,Roig C,Diaz A,Blanca J M,Garcia-MasJ,Gómez-Guillamón M L,López-SeséAⅠ,Lázaro A,Monforte AJ,Pico B. SNP genotyping in melons:genetic variation,populationstructure,and linkage disequilibrium[J]. Theoretical&AppliedGenetics,2013,126(5):1285-1304
[27] Tajima F. Statistical method for testing the neutral mutationhypothesis by DNA polymorphism[J]. Genetics,1989,123(3):585-595
[28] Fu Y X,Li W H. Statistical tests of neutrality of mutations[J].Genetics,1993,133(3):693-709
[29] Tsai C J,Harding S A,Tschaplinski T J,Lindroth R L,Yuan Y.Genome-wide analysis of the structural genes regulating defensephenylpropanoid metabolism in populus[J]. New Phytologist,2006,172(1):47-62
[30] Martz F,Maury S,Pinon G,Legrand M. c DNA cloning,substratespecificity and expression study of tobacco caffeoyl-CoA 3-O-methyltransferase,a lignin biosynthetic enzyme[J]. Plant MolecularBiology,1998,36(3):427-437
[31]赵华燕.利用反义RNA技术进行木质素生物合成调控的研究[D].北京:中国科学院研究生院,2004:46-54
[32]李玉岭,周厚君,林二培,黄华宏,徐莉莉,童再康.光皮桦BlSPL1转录因子基因的克隆、表达及单核苷酸多态性分析[J].林业科学,2013,49(9):52-61
[33]徐煲铧,杨晓慧,李百炼,张志毅,张德强.毛白杨纤维素合酶基因PtCesA4的克隆、表达及单核苷酸多态性分析[J].林业科学,2009,45(5):1-10
[34]龚莺,陈虹君,许在恩,郭小勤.竹类植物Dwarf 14(D14)基因的多态性分析[J].核农学报,2018,32(1):48-57
[35]郭琦,王保垒,王博文,李百炼,张德强.毛白杨PtDREB2A基因的克隆、表达及单核苷酸多态性分析[J].林业科学,2011,47(4):49-56
[36] Zhu Y L,Song Q J,Hyten D L,Van Tassell C P,Matukumalli LK,Grimm D R,Hyatt S M,Fickus E W,Young N D,Cregan P B.Single-nucleotide polymorphisms in soybean[J]. Genetics,2003,163(3):1123-1134
[37] Guerra F P,Wegrzyn J L,Sykes R,Davis M F,Stanton B J,NealeD B. Association genetics of chemical wood properties in blackpoplar(Populus nigra)[J]. New Phytologist,2013,197(1):162-176
[38] Chen J,Xie J,Chen B,Quan M,Li Y,Li B,Zhang D. Geneticvariations and miRNA-target interactions contribute to naturalphenotypic variations in populus[J]. New Phytologist,2016,212(1):150-160
[39] Dillon S K,Nolan M F,Wu H,Southerton S G,Matheson C.Association genetics reveal candidate gene SNPs affecting woodproperties in Pinus radiata[J]. Australian Forestry,2010,73(3):185-190
[2]李潞滨,刘蕾,何聪芬,董银卯,彭镇华.木质素生物合成关键酶基因的研究进展[J].分子植物育种,2007,5(S1):45-51
[3] Hao Z,Mohnen D. A review of xylan and lignin biosynthesis:foundation for studying Arabidopsis irregular xylem mutants withpleiotropic phenotypes[J]. Critical Reviews in Biochemistry&Molecular Biology,2014,49(3):212-241
[4] Wang H,Yu Y,Fu C X,Zhou G K,Gao H H. Progress of a keyenzyme caffeoyl-CoA 3-O-methyltransferase in lignin biosynthesis[J]. Genomics&Applied Biology,2014,33(2):458-466
[5] Hu D,Liu X B,She H Z,Hu D,Liu X B,She H Z,Gao Z,RuanR,Wu D Q,Yi Z L. The lignin synthesis related genes and lodgingresistance of Fagopyrum esculentum[J]. Biologia Plantarum,2017,61(1):138-146
[6] Xu R X,Zhao Y,Gao S,Zhang Y Y,Li D D,Lou H X,Cheng AX. Functional characterization of a plastidal cation-dependent O-methyltransferase from the liverwort Plagiochasma appendiculatum[J]. Phytochemistry,2015,8(2):33-41
[7] Do C T,Pollet B,Thévenin J,Sibout R,Denoue D,Barrière Y,Catherine L, Jouanin L. Both caffeoyl coenzyme a 3-O-methyltransferase 1 and caffeic acid O-methyltransferase 1 areinvolved in redundant functions for lignin,flavonoids and sinapoylmalate biosynthesis in Arabidopsis[J]. Planta,2007,226(5):1117-1129
[8] Li X Y,Chen W J,Zhao Y,Xiang Y,Jiang H Y,Zhu S W,ChengB J. Downregulation of caffeoyl-CoA O-methyltransferase(CCoAOMT)by RNA interference leads to reduced ligninproduction in maize straw[J]. Genetics&Molecular Biology,2013,36(4):540-546
[9] Zhong R,Morrison W H,Himmelsbach D S,Poole F L,Ye Z H.Essential role of caffeoyl coenzyme a O-methyltransferase in ligninbiosynthesis in woody poplar plants[J]. Plant Physiology,2000,124(2):563-578
[10] Liu S J,Huang Y H,He C J,Fang C,Zhang Y W. Cloning,bioinformatics and transcriptional analysis of caffeoyl coenzyme a 3-O-methyltransferase in switchgrass under abiotic stress[J]. Journalof Integrative Agriculture,2016,15(3):636-649
[11] FornaléS, Rencoret J, García-calvo L, Encina A, Rigau J,Gutiérrez A,Río J C D,Caparros-Ruiz D. Changes in cell wallpolymers and degradability in maize mutants lacking 3'-and 5'-O-methyltransferases involved in lignin biosynthesis[J]. Plant&CellPhysiology,2016,58(2):240-255
[12]杨业斌.光皮桦的经济性状及栽培技术[J].湖南林业科技,1990(4):20-21
[13]黄华宏.基于转录组测序的光皮桦应拉木形成分子机制研究[D].杭州:浙江大学,2012:21-28
[14] Huang H H,Jiang C,Tong Z K,Cheng L J,Zhu M Y,Lin E P.Eight distinct cellulose synthase catalytic subunit genes from Betulaluminifera are associated with primary and secondary cell wallbiosynthesis[J]. Cellulose,2014,21(4):2183-2198
[15]江成.光皮桦BlOFPs基因的克隆及其功能研究[D].杭州:浙江农林大学,2014:21-22,50
[16]赵宏亮,陈凯,张强,徐正进,黎志康.基于连锁不平衡水稻源库相关性状的关联分析[J].核农学报,2015,29(4):674-684
[17]巩琛锐,王璐,杜庆章,张德强.毛白杨S-腺苷高半胱氨酸水解酶基因PtSAHHA的克隆、表达及SNP关联分析[J].林业科学,2013,49(11):67-76
[18] Colasuonno P,Gadaleta A,Giancaspro A,Nigro D,Giove S,Incerti O, Mangini G, Signorile A, Simeone R, Blanco A.Development of a high-density SNP-based linkage map anddetection of yellow pigment content QTLs in durum wheat[J].Molecular Breeding,2014,34(4):1563-1578
[19]何辉,楼雄珍,林二培,俞友明,童再康,黄华宏.光皮桦应拉木的显微特征及其形成早期内源激素分布[J].林业科学,2016,52(10):38-44
[20]张俊红,黄华宏,童再康,程龙军,梁跃龙,陈奕良.光皮桦6个南方天然群体的遗传多样性[J].生物多样性,2010,18(3):233-240
[21] Kumar S,Stecher G,Tamura K. MEGA7:molecular evolutionarygenetics analysis version 7. 0 for bigger datasets[J]. MolecularBiology and Evolution,2016,33(7):1870-1874
[22] Librado P, Rozas J. DnaSP v5:a software for comprehensiveanalysis of DNA polymorphism data[J]. Bioinformatics,2009,25(11):1451-1452
[23] Joshi C P,ChiangⅤL. Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases[J]. PlantMolecular Biology,1998,37(4):663
[24] Mizrachi E,MaloneyⅤJ,Silberbauer J,Hefer C A,Berger D K,Mansfield S D, Myburg A A. Investigating the molecularunderpinnings underlying morphology and changes in carbonpartitioning during tension wood formation in Eucalyptus[J]. NewPhytologist,2015,206(4):1351-1363
[25] Chang S S,Salmén L,Olsson A M,Clair B. Deposition andorganization of cell wall polymers during maturation of poplar tensionwood by FTIR microspectroscopy[J]. Planta,2014,239(1):243-254
[26] Esteras C,Formisano G,Roig C,Diaz A,Blanca J M,Garcia-MasJ,Gómez-Guillamón M L,López-SeséAⅠ,Lázaro A,Monforte AJ,Pico B. SNP genotyping in melons:genetic variation,populationstructure,and linkage disequilibrium[J]. Theoretical&AppliedGenetics,2013,126(5):1285-1304
[27] Tajima F. Statistical method for testing the neutral mutationhypothesis by DNA polymorphism[J]. Genetics,1989,123(3):585-595
[28] Fu Y X,Li W H. Statistical tests of neutrality of mutations[J].Genetics,1993,133(3):693-709
[29] Tsai C J,Harding S A,Tschaplinski T J,Lindroth R L,Yuan Y.Genome-wide analysis of the structural genes regulating defensephenylpropanoid metabolism in populus[J]. New Phytologist,2006,172(1):47-62
[30] Martz F,Maury S,Pinon G,Legrand M. c DNA cloning,substratespecificity and expression study of tobacco caffeoyl-CoA 3-O-methyltransferase,a lignin biosynthetic enzyme[J]. Plant MolecularBiology,1998,36(3):427-437
[31]赵华燕.利用反义RNA技术进行木质素生物合成调控的研究[D].北京:中国科学院研究生院,2004:46-54
[32]李玉岭,周厚君,林二培,黄华宏,徐莉莉,童再康.光皮桦BlSPL1转录因子基因的克隆、表达及单核苷酸多态性分析[J].林业科学,2013,49(9):52-61
[33]徐煲铧,杨晓慧,李百炼,张志毅,张德强.毛白杨纤维素合酶基因PtCesA4的克隆、表达及单核苷酸多态性分析[J].林业科学,2009,45(5):1-10
[34]龚莺,陈虹君,许在恩,郭小勤.竹类植物Dwarf 14(D14)基因的多态性分析[J].核农学报,2018,32(1):48-57
[35]郭琦,王保垒,王博文,李百炼,张德强.毛白杨PtDREB2A基因的克隆、表达及单核苷酸多态性分析[J].林业科学,2011,47(4):49-56
[36] Zhu Y L,Song Q J,Hyten D L,Van Tassell C P,Matukumalli LK,Grimm D R,Hyatt S M,Fickus E W,Young N D,Cregan P B.Single-nucleotide polymorphisms in soybean[J]. Genetics,2003,163(3):1123-1134
[37] Guerra F P,Wegrzyn J L,Sykes R,Davis M F,Stanton B J,NealeD B. Association genetics of chemical wood properties in blackpoplar(Populus nigra)[J]. New Phytologist,2013,197(1):162-176
[38] Chen J,Xie J,Chen B,Quan M,Li Y,Li B,Zhang D. Geneticvariations and miRNA-target interactions contribute to naturalphenotypic variations in populus[J]. New Phytologist,2016,212(1):150-160
[39] Dillon S K,Nolan M F,Wu H,Southerton S G,Matheson C.Association genetics reveal candidate gene SNPs affecting woodproperties in Pinus radiata[J]. Australian Forestry,2010,73(3):185-190
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |