In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL 1 was constructed. The recombinant was identified by three restriction endonucleases, then...In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL 1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL 1 was transformed into expression host M15 （pREP4） and induced by isopropyl-a-D-thiogalactoside （IPTG） to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol＇L-1 IPTG induction as the expression temperature declined from 37 to 28℃. The 6-His tag facilitates affinity binding to Ni^2＋-nitrolotriacetic acid （NTA） and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose coupled with Ni^2＋-NTA affinity chromatography. The optimal substrate for Pt4CL 1 was 4-coumarate.展开更多
4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the me...4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen （Salvia miltiorrhiza Bunge）, a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs （Sm4CL1 and Sm4CL2） encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction （RT-PCR） with degenerate primers followed by 5′/3′rapid amplification of cDNA ends （RACE） （Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164）. Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were （72.20±4.10） and （6.50±1.45） μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.展开更多
目的:对珊瑚菜(Glehnia littoralis)4-香豆酸:辅酸A连接酶(4-Coumarate:Coenzyme A Ligase,Gl4CL)基因编码区进行克隆及序列分析。方法:本研究在前期珊瑚菜高通量测序的基础上,利用c DNA末端快速扩增(Rapid Amplification of c DNA Ends...目的:对珊瑚菜(Glehnia littoralis)4-香豆酸:辅酸A连接酶(4-Coumarate:Coenzyme A Ligase,Gl4CL)基因编码区进行克隆及序列分析。方法:本研究在前期珊瑚菜高通量测序的基础上,利用c DNA末端快速扩增(Rapid Amplification of c DNA Ends,RACE)方法对Gl4CL基因全长c DNA序列进行克隆。对Gl4CL蛋白进行理化性质、蛋白二级结构及三维结构预测分析。利用实时荧光定量PCR方法检测Gl4CL基因在珊瑚菜的根、叶中的表达情况。结果:Gl4CL基因cDNA序列全长1 951 bp,编码544个氨基酸,其中开放阅读框1 635 bp,5′非编码区153 bp,3′非编码区163 bp。生物信息学分析表明,Gl4CL蛋白大小约为59.481 k Da,等电点为8.20。Gl4CL基因在珊瑚菜的根和叶均存在表达,且在根中表达量显著高于叶。结论:本研究为进一步开展Gl4CL基因功能和遗传调控研究奠定基础,通过深入探讨Gl4CL基因的表达调控与木质素、植株生长表型等关系,有望获得抗病虫害、抗逆性强的北沙参高产优质品系。展开更多
文摘In order to investigate the enzymatic properties of the 4CL1 of Populus tomentosa, the recombinant expression vector pQE31-4CL 1 was constructed. The recombinant was identified by three restriction endonucleases, then the vector pQE31-4CL 1 was transformed into expression host M15 （pREP4） and induced by isopropyl-a-D-thiogalactoside （IPTG） to express 60 kD fused protein Pt4CL1. The biologically active Pt4CL1, expressed as soluble protein, was achieved with 0.6 mmol＇L-1 IPTG induction as the expression temperature declined from 37 to 28℃. The 6-His tag facilitates affinity binding to Ni^2＋-nitrolotriacetic acid （NTA） and enables one-step purification to acquire the molecular SDS-PAGE electrophoresis purity of the active 4CL1 protein by agarose coupled with Ni^2＋-NTA affinity chromatography. The optimal substrate for Pt4CL 1 was 4-coumarate.
基金Supported by the National Natural Science Foundation of China （30300447）.The authors thank Dr Chen Yongning （China Innovation Centre for Drug Development, HK） for useful suggestions and support. The authors also thank to Dr Fanya Zeng and Miss Charis Chan （Department of Zoology, University of Hong Kong） for technical assistance.
文摘4-Coumarate ： coenzyme A Ilgase （4CL） Is one of the key enzymes In phenylpropanoid metabolism leading to series of phenollcs, Including water-soluble phenolic acids, which are important compounds determining the medicinal quality of Danshen （Salvia miltiorrhiza Bunge）, a traditional Chinese medicinal herb. To Investigate the function of 4CL in the biosynthesis of water-soluble phenolic acid in Danshen, we have cloned two cDNAs （Sm4CL1 and Sm4CL2） encoding divergent 4CL members by applying nested reverse transcrlptlon-polymerase chain reaction （RT-PCR） with degenerate primers followed by 5′/3′rapid amplification of cDNA ends （RACE） （Note, these sequence data have been submitted to the GenBank database under accession numbers AY237163 and AY237164）. Either of the coding regions was inserted into a pRSET vector and a kinetic assay was performed with purified recombinant proteins. The substrate utilization profile of Sm4CL1 was distinct from that of Sm4CL2. The Km values of Sm4CL1 and Sm4CL2 to 4-coumarlc acid were （72.20±4.10） and （6.50±1.45） μmol/L, respectively. These results, In conjunction with Northern blotting and other information, imply that Sm4CL2 may play an Important role in the biosynthesis of watersoluble phenolic compounds, whereas Sm4CL1 may play a minor role in the pathway. Southern blotting analysis suggested that both Sm4CL1 and Sm4CL2 genes are present as a single copy and are located at different sites In the genome.
文摘目的:对珊瑚菜(Glehnia littoralis)4-香豆酸:辅酸A连接酶(4-Coumarate:Coenzyme A Ligase,Gl4CL)基因编码区进行克隆及序列分析。方法:本研究在前期珊瑚菜高通量测序的基础上,利用c DNA末端快速扩增(Rapid Amplification of c DNA Ends,RACE)方法对Gl4CL基因全长c DNA序列进行克隆。对Gl4CL蛋白进行理化性质、蛋白二级结构及三维结构预测分析。利用实时荧光定量PCR方法检测Gl4CL基因在珊瑚菜的根、叶中的表达情况。结果:Gl4CL基因cDNA序列全长1 951 bp,编码544个氨基酸,其中开放阅读框1 635 bp,5′非编码区153 bp,3′非编码区163 bp。生物信息学分析表明,Gl4CL蛋白大小约为59.481 k Da,等电点为8.20。Gl4CL基因在珊瑚菜的根和叶均存在表达,且在根中表达量显著高于叶。结论:本研究为进一步开展Gl4CL基因功能和遗传调控研究奠定基础,通过深入探讨Gl4CL基因的表达调控与木质素、植株生长表型等关系,有望获得抗病虫害、抗逆性强的北沙参高产优质品系。
