柠条锦鸡儿木质素合成关键基因CkCOMT的克隆及表达载体构建
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摘要
为了提高柠条锦鸡儿的抗非生物胁迫能力和饲草价值,利用分子生物学和生物信息学方法,克隆并分析柠条锦鸡儿木质素合成关键基因CkCOMT。在获得409 bp中间片段基础上,通过RACE技术,拼接获得基因全长1 378 bp,其中包括75 bp的5'非编码区,205 bp的3'非编码区,polyA (12)尾,ORF为1 098 bp,编码365个氨基酸。经预测分析,CkCOMT蛋白分子量为39.95 k D,属稳定的疏水性蛋白,二级结构以无规则卷曲为主。在该基因与豆科其他植物及模式植物的氨基酸序列进化树分析中,CkCOMT与苜蓿及大豆的亲缘关系最近。在此基础上,CkCOMT基因全长片段与p CanG-HA线性载体连接,成功构建pCanG-CkCOMT-HA重组质粒。该研究为下一步分析CkCOMT基因在木质素合成和代谢途径中的调控作用奠定了基础,同时,也为提高柠条锦鸡儿生存能力和经济价值提供了理论条件。
In order to improve abiotic stress tolerance and forage value of Caragana korshinskii, the lignin synthesis key gene CkCOMT was cloned and analyzed under the help of molecular biological and bioinformatics methods.The core sequence was 409 bp fragment, by means of RACE, and we get 1 378 bp total sequence which included75 bp 5' noncoding region, 205 bp 3' noncoding region and polyA(12) sequence. Its ORF was 1 098 bp and it encoded 365 amino acids. After predictive analysis, CkCOMT protein molecular weight was 39.95 kD, which was a stable hydrophobic protein, and most of secondary structures were irregular curls. In the phylogenetic analysis of the amino acid sequence of this gene and other plants of the leguminous family and model plants, it had the closest genetic relationship with Medicago sativa and Glycine max. On this basis, the pCanG-CkCOMT-HA recombinant plasmid was successfully constructed by the CkCOMT full sequence linked to pCanG-HA linear expression vector.This study could lay the foundation for the analysis of the regulation of CkCOMT gene in lignin synthetic and metabolic pathways. At the same time, it could offer theoretical conditions for enhancing the survival and economic value of Caragana korshinskii.
In order to improve abiotic stress tolerance and forage value of Caragana korshinskii, the lignin synthesis key gene CkCOMT was cloned and analyzed under the help of molecular biological and bioinformatics methods.The core sequence was 409 bp fragment, by means of RACE, and we get 1 378 bp total sequence which included75 bp 5' noncoding region, 205 bp 3' noncoding region and polyA(12) sequence. Its ORF was 1 098 bp and it encoded 365 amino acids. After predictive analysis, CkCOMT protein molecular weight was 39.95 kD, which was a stable hydrophobic protein, and most of secondary structures were irregular curls. In the phylogenetic analysis of the amino acid sequence of this gene and other plants of the leguminous family and model plants, it had the closest genetic relationship with Medicago sativa and Glycine max. On this basis, the pCanG-CkCOMT-HA recombinant plasmid was successfully constructed by the CkCOMT full sequence linked to pCanG-HA linear expression vector.This study could lay the foundation for the analysis of the regulation of CkCOMT gene in lignin synthetic and metabolic pathways. At the same time, it could offer theoretical conditions for enhancing the survival and economic value of Caragana korshinskii.
引文
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Carocha V.,Soler M.,Hefer C.,Cassanwang H.,Fevereiro P.,Myburg A.A.,Paiva J.A.,and Grima-Pettenati J.,2015,Genome-wide analysis of the lignin toolbox of Eucalyptus grandis,New Phytol.,206(4):1297-1313
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Fan L.,2013,Bioinformatic and functional analysis of mulberrygenes involved in lignin biosynthesis,Thesis for M.S.,Southwest University,Supervisor:He N.J.,pp.1-11(范丽,2013,桑树木质素合成基因的生物信息和功能分析,硕士学位论文,西南大学,导师:何宁佳,pp.1-11)
Gallego-Giraldo L.,Shadle G.,Shen H.,Barros-Rios J.,Corrales S.F.,Wang H.Z.,and Dixon R.A.,2016,Combining enhanced biomass density with reduced lignin level for improved forage quality,Plant Biotechnol.J.,14(3):895-904
Hu D.,Liu X.B.,She H.Z.,Gao Z.,Ruan R.W.,Wu D.Q.,and Yi Z.L.,2017,The lignin synthesis related genes and lodging resistance of Fagopyrum esculentum,Biol.Plantarum,61(1):138-146
Li Y.,Gu H.,and Qi C.K.,2013,Expresion of ligin biothesis key genes of F5H,4CL and COMT on lodging resistance of Brassica napus L.,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Science),35(1):24-28(李扬,顾慧,戚存扣,2013,甘蓝型油菜木质素合成关键基因F5H、4CL和COMT的定量表达,中国油料作物学报,35(1):24-28)
Li W.,Lu J.,Lu K.,Yuan J.L.,Huang J.H.,Du H.,and Li J.N.,2016,Cloning and phylogenetic analysis of Brassica napus L.caffeic acid o-methyltransferase 1 gene family and its expression pattern under drought stress,PLoS One,11(11):165975
Schwede T.,Kopp J.,Guex N.,and Peitsch M.C.,2003,SWISS-MODEL:an automated protein homology-modeling server,Nucleic Acids Res.,31(13):3381-3385
Somerville C.,Bauer S.,Brininstool G.,Facette M.,Hamann T.,Milne J.,Osborne E.,Paredez A.,Persson S.,Raab T.,Vorwerk S.,Youngs H..,2004,Toward a systems approach to understanding plant cell walls,Science,306(5705):2206-2211
Vanholme R.,Morreel K.,Ralph J.,and Boerjan W.,2008,Lignin engineering,Curr.Opin.Plant.Biol.,11(3):278-285
Vélez-Bermúdez I.C.,Salazarhenao J.E.,FornaléS.,Lópezvidriero I.,Franco-Zorrilla J.M.,Grotewold E.,Gray J.,Solano R.,Schmidt W.,Pagés M.,Riera M.,and Caparros-Ruiz D.,2015,A MYB/ZML complex regulates wound-induced lignin genes in maize,Plant Cell,27(11):3245-3259
Weng J.K.,and Chapple C.,2010,The origin and evolution of lignin biosynthesis,New Phytol.,187(2):273-285
Yin J.,2013,Gene mapping transcriptome sequencing and cloning of two ligin biosynthetic genes of a stemlodging mutant in soybean,Thesis for M.S.,Nanjing Agricultural University,Supervisor:Zhao T.J.,pp.1-16(殷静,2013,大豆茎倒伏突变体基因定位、转录组测序及2个木质素合成基因的克隆研究,硕士学位论文,南京农业大学,导师:赵团结,pp.1-16)
Baxter H.L.,Mazarei M.,Fu C.,Cheng Q.K.,Turner G.B.,Sykes R.W.,Windham M.T.,Davis M.F.,Dixon R.A.,Wang Z.Y.,and Stewart C.N.,2016,Time course field analysis of CO-MT-downregulated switchgrass:lignification,recalcitrance,and rust susceptibility,Bioenerg.Res.,9(4):1087-1100
Besseau S.,Hoffmann L.,Geoffroy P.,Lapierre G.,Pollet B.,and Legrand M.,2007,Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth,Plant Cell,19(1):148-162
Boerjan W.,Ralph J.,and Baucher M.,2003,Lignin biosynthesis,Ann.Rev.Plant Biol.,54:519-546
Carocha V.,Soler M.,Hefer C.,Cassanwang H.,Fevereiro P.,Myburg A.A.,Paiva J.A.,and Grima-Pettenati J.,2015,Genome-wide analysis of the lignin toolbox of Eucalyptus grandis,New Phytol.,206(4):1297-1313
Coleman H.D.,Samuels A.L.,Guy R.D.,and Mansfield S.D.,2008,Perturbed lignification impacts tree growth in hybrid poplar-a function of sink strength,vascular integrity,and photosynthetic assimilation,Plant Physiol.,148(3):1229-1237
Fan L.,2013,Bioinformatic and functional analysis of mulberrygenes involved in lignin biosynthesis,Thesis for M.S.,Southwest University,Supervisor:He N.J.,pp.1-11(范丽,2013,桑树木质素合成基因的生物信息和功能分析,硕士学位论文,西南大学,导师:何宁佳,pp.1-11)
Gallego-Giraldo L.,Shadle G.,Shen H.,Barros-Rios J.,Corrales S.F.,Wang H.Z.,and Dixon R.A.,2016,Combining enhanced biomass density with reduced lignin level for improved forage quality,Plant Biotechnol.J.,14(3):895-904
Hu D.,Liu X.B.,She H.Z.,Gao Z.,Ruan R.W.,Wu D.Q.,and Yi Z.L.,2017,The lignin synthesis related genes and lodging resistance of Fagopyrum esculentum,Biol.Plantarum,61(1):138-146
Li Y.,Gu H.,and Qi C.K.,2013,Expresion of ligin biothesis key genes of F5H,4CL and COMT on lodging resistance of Brassica napus L.,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Science),35(1):24-28(李扬,顾慧,戚存扣,2013,甘蓝型油菜木质素合成关键基因F5H、4CL和COMT的定量表达,中国油料作物学报,35(1):24-28)
Li W.,Lu J.,Lu K.,Yuan J.L.,Huang J.H.,Du H.,and Li J.N.,2016,Cloning and phylogenetic analysis of Brassica napus L.caffeic acid o-methyltransferase 1 gene family and its expression pattern under drought stress,PLoS One,11(11):165975
Schwede T.,Kopp J.,Guex N.,and Peitsch M.C.,2003,SWISS-MODEL:an automated protein homology-modeling server,Nucleic Acids Res.,31(13):3381-3385
Somerville C.,Bauer S.,Brininstool G.,Facette M.,Hamann T.,Milne J.,Osborne E.,Paredez A.,Persson S.,Raab T.,Vorwerk S.,Youngs H..,2004,Toward a systems approach to understanding plant cell walls,Science,306(5705):2206-2211
Vanholme R.,Morreel K.,Ralph J.,and Boerjan W.,2008,Lignin engineering,Curr.Opin.Plant.Biol.,11(3):278-285
Vélez-Bermúdez I.C.,Salazarhenao J.E.,FornaléS.,Lópezvidriero I.,Franco-Zorrilla J.M.,Grotewold E.,Gray J.,Solano R.,Schmidt W.,Pagés M.,Riera M.,and Caparros-Ruiz D.,2015,A MYB/ZML complex regulates wound-induced lignin genes in maize,Plant Cell,27(11):3245-3259
Weng J.K.,and Chapple C.,2010,The origin and evolution of lignin biosynthesis,New Phytol.,187(2):273-285
Yin J.,2013,Gene mapping transcriptome sequencing and cloning of two ligin biosynthetic genes of a stemlodging mutant in soybean,Thesis for M.S.,Nanjing Agricultural University,Supervisor:Zhao T.J.,pp.1-16(殷静,2013,大豆茎倒伏突变体基因定位、转录组测序及2个木质素合成基因的克隆研究,硕士学位论文,南京农业大学,导师:赵团结,pp.1-16)
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