长春碱生物合成途径中关键酶基因的克隆与生物信息学分析
|
摘要
以长春花幼苗的叶片为材料,通过RT-PCR克隆关键酶基因CDS序列,利用在线软件ExPASy ProtParam、SignalP 4.1、TMHMM V.2.0、TargetP 1.1分析蛋白理化性质、蛋白信号肽、蛋白跨膜区以及亚细胞定位,在线工具Conserved Domain、SOPMA预测蛋白保守结构域和二级结构,MEGA6.0软件构建蛋白系统进化树,为后续的TIA合成提供理论研究依据。结果表明,STR、TDC、和ORCA3关键酶基因的CDS序列大小分别为1 059、1 503、612 bp;生物信息学分析表明,STR、TDC、和ORCA3关键酶定位于细胞不同区室,为酸性蛋白,无信号肽,除STR外无跨膜区,由α-螺旋、延伸链、β-转角和无规则卷曲等二级结构组成;除TDC外均包含高度保守的结构域;聚类分析表明其与小蔓长春花、罗芙木和中果咖啡具有较高的相似性,较近的亲缘关系和遗传距离,该结果可为明确该基因在TIA合成代谢途径中的重要作用奠定基础。
The leaves of Catharanthus roseus L.were used as materials to clone the gene CDS sequence by RT-PCR.The online software ExPASy ProtParam,SignalP 4.1,TMHMM V.2.0,TargetP 1.1 were used to analyze the physical and chemical properties of proteins,protein signal peptides and proteins transmembrane region and subcellular localization.Online tool Conserved Domain,SOPMA predicted protein conserved domain and secondary structure,MEGA6.0 software constructed protein system phylogenetic tree,in order to provide theoretical basis for the subsequent TIA synthesis.The results showed that the CDS sequences of STR,TDC and ORCA3 key enzyme genes were obtained,fragment sizes were 1 059 bp,1 503 bp,and 612 bp,respectively.Bioinformatics analysis showed that the key enzymes of STR,TDC and ORCA3 were localized in different compartments of cells.It is an acidic protein,no signal peptide,and has no transmembrane region except STR.It consists of secondary structures such as α-helix,extended chain,β-turn and random coil,all of them contain highly conserved domains except TDC. Cluster analysis showed that it had higher similarity with the Vinca minor L.,Rauvolfia verticillata(Lour) Baill and Coffee canephora,and the closer genetic relationship and genetic distance,these results may lay a foundation for the important role of the gene in TIA biosynthesis pathway.
The leaves of Catharanthus roseus L.were used as materials to clone the gene CDS sequence by RT-PCR.The online software ExPASy ProtParam,SignalP 4.1,TMHMM V.2.0,TargetP 1.1 were used to analyze the physical and chemical properties of proteins,protein signal peptides and proteins transmembrane region and subcellular localization.Online tool Conserved Domain,SOPMA predicted protein conserved domain and secondary structure,MEGA6.0 software constructed protein system phylogenetic tree,in order to provide theoretical basis for the subsequent TIA synthesis.The results showed that the CDS sequences of STR,TDC and ORCA3 key enzyme genes were obtained,fragment sizes were 1 059 bp,1 503 bp,and 612 bp,respectively.Bioinformatics analysis showed that the key enzymes of STR,TDC and ORCA3 were localized in different compartments of cells.It is an acidic protein,no signal peptide,and has no transmembrane region except STR.It consists of secondary structures such as α-helix,extended chain,β-turn and random coil,all of them contain highly conserved domains except TDC. Cluster analysis showed that it had higher similarity with the Vinca minor L.,Rauvolfia verticillata(Lour) Baill and Coffee canephora,and the closer genetic relationship and genetic distance,these results may lay a foundation for the important role of the gene in TIA biosynthesis pathway.
引文
[1]ALMAGRO L,FERNNDEZ-PREZ F,PEDREO M A.Indole alkaloids from Catharanthus roseus:bioproduction and their effect on human health[J].Molecules,2015,20(2):2973.
[2]ZHU J,WANG M,WEN W,et al.Biosynthesis and regulation of terpenoid indole alkaloids in Catharanthus roseus[J].Pharmacogn Rev,2015,9(17):24.
[3]PASQUALI G,PORTO D D,FETT-NETO A G.Metabolic engineering of cell culturesversus whole plant complexityin production of bioactive monoterpene indole alkaloids:recent progress relatedto old dilemma[J].Journal of Bioscience and Bioengineering,2006,101(4):287-296.
[4]HALLARD D,VAN DER HEIJDEN R,Verpoorte R,et al.Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding[J].Plant Cell Rep,1997,17,50.
[5]CHAVADEJ S,BRISSON N,MCNEIL J N.Redirection of tryptophan leads to production of low indole glucosinolatecanola[J].Proc Natl Acad Sci USA,1994,91:2166.
[6]PEEBLES C A,HUGHES E H,SHANKS J V,et al.Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time[J].Metab Eng,2009,11(2):76.
[7]VANDER FITS L,MEMELINK J.Memelink,ORCA3,a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[J].Science,2000,289(5477):295.
[8]韩梅,赵博,安志刚,等.长春花萜类吲哚生物碱生物合成途径中重要酶(DXR、SLS、G10H、STR)基因的克隆与表达[J].植物研究,2007(5):564-568.
[9]陈士林,宋经元.本草基因组学[J].中国中药杂志,2016,41(21):3381.
[10]CHEN S L,SONG J Y,SUN C,et al.Herbal genomics:examining the biology of traditional medicines[J].Science,2015,347(增刊1):27.
[11]匡雪君,王彩霞,邹丽秋,等.长春花萜类吲哚生物碱生物合成与调控研究[J].中国中药杂志,2016,41(22):4129-4137.
[2]ZHU J,WANG M,WEN W,et al.Biosynthesis and regulation of terpenoid indole alkaloids in Catharanthus roseus[J].Pharmacogn Rev,2015,9(17):24.
[3]PASQUALI G,PORTO D D,FETT-NETO A G.Metabolic engineering of cell culturesversus whole plant complexityin production of bioactive monoterpene indole alkaloids:recent progress relatedto old dilemma[J].Journal of Bioscience and Bioengineering,2006,101(4):287-296.
[4]HALLARD D,VAN DER HEIJDEN R,Verpoorte R,et al.Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding[J].Plant Cell Rep,1997,17,50.
[5]CHAVADEJ S,BRISSON N,MCNEIL J N.Redirection of tryptophan leads to production of low indole glucosinolatecanola[J].Proc Natl Acad Sci USA,1994,91:2166.
[6]PEEBLES C A,HUGHES E H,SHANKS J V,et al.Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time[J].Metab Eng,2009,11(2):76.
[7]VANDER FITS L,MEMELINK J.Memelink,ORCA3,a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[J].Science,2000,289(5477):295.
[8]韩梅,赵博,安志刚,等.长春花萜类吲哚生物碱生物合成途径中重要酶(DXR、SLS、G10H、STR)基因的克隆与表达[J].植物研究,2007(5):564-568.
[9]陈士林,宋经元.本草基因组学[J].中国中药杂志,2016,41(21):3381.
[10]CHEN S L,SONG J Y,SUN C,et al.Herbal genomics:examining the biology of traditional medicines[J].Science,2015,347(增刊1):27.
[11]匡雪君,王彩霞,邹丽秋,等.长春花萜类吲哚生物碱生物合成与调控研究[J].中国中药杂志,2016,41(22):4129-4137.
© 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 |