广藿香PTS基因的克隆及CPEC法构建其过表达载体pRI101-PTS
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摘要
目的克隆广藿香中广藿香醇合酶(PTS)基因的cDNA序列,并构建其pRI101-PTS植物表达载体。方法利用逆转录聚合酶链式反应(RT-PCR)获得PTS基因的cDNA序列,利用生物信息学软件对T-A克隆后的序列进行分析,预测其编码蛋白的理化性质、结构和功能。以环形聚合酶延伸克隆(CPEC)构建pRI101-PTS过表达载体。结果 PTS基因的开放阅读框全长1 659 bp,编码552个氨基酸。该蛋白分子量为64.1 ku,等电点为5.39,含有DDxxD保守序列,具有2个倍半萜合酶结构域,C末端结构域268个氨基酸残基可能涉及线性萜烯的环化,成功构建了pRI101-PTS过表达载体。结论 PTS的克隆、植物过表达载体的构建及生物信息学分析为其他倍半萜类合成酶基因的发现和研究提供了参考,也为进一步研究其生物合成调控机制甚至代谢工程奠定基础。
Objective To clone the Patchoulol synthase(PTS) gene from total RNA of Pogostemon cablin(Blanco) Benth. and construct the overexpression vector pRI101-PTS. Methods The full length cDNA of PTS was obtained by reverse transcription-PCR(RT-PCR). After T-A cloning,the sequence similarity and homology were analyzed,while the structure and function of the coding protein were predicted by bioinformatics method. The pRI101-PTS overexpression vector was constructed by using circular polymerase extension cloning(CPEC). Results The PTS gene was cloned,which contained an ORF of 1 659 bp and encoded 552 amino acids. This protein had a molecular weight of 64.1 ku,an isoelectric point of 5.39,a conserved DDxxD motifs and two sesquiterpene synthase domains. The C-terminal domain which contained 268 amino acid residues,might be involved in the cyclization of linear terpenes and constructed the overexpression vector pRI101-PTS successfully. Conclusion The cloning of PTS gene,construction of overexpression vector and bioinformatics analysis provide reference for the discovery and research of other sesquiterpene synthase genes,and lay the foundation for further research on its biosynthesis,regulation mechanism and even metabolic engineering.
Objective To clone the Patchoulol synthase(PTS) gene from total RNA of Pogostemon cablin(Blanco) Benth. and construct the overexpression vector pRI101-PTS. Methods The full length cDNA of PTS was obtained by reverse transcription-PCR(RT-PCR). After T-A cloning,the sequence similarity and homology were analyzed,while the structure and function of the coding protein were predicted by bioinformatics method. The pRI101-PTS overexpression vector was constructed by using circular polymerase extension cloning(CPEC). Results The PTS gene was cloned,which contained an ORF of 1 659 bp and encoded 552 amino acids. This protein had a molecular weight of 64.1 ku,an isoelectric point of 5.39,a conserved DDxxD motifs and two sesquiterpene synthase domains. The C-terminal domain which contained 268 amino acid residues,might be involved in the cyclization of linear terpenes and constructed the overexpression vector pRI101-PTS successfully. Conclusion The cloning of PTS gene,construction of overexpression vector and bioinformatics analysis provide reference for the discovery and research of other sesquiterpene synthase genes,and lay the foundation for further research on its biosynthesis,regulation mechanism and even metabolic engineering.
引文
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[13] 万兵,闫杰,季明春,等.一种优化的PCR方法——降落PCR扩增目的基因[J].江苏临床医学杂志,2002,6(2):127-129.
[14] 马转转,庞潇卿,谌容,等.萜类化合物生物合成途径中关键酶的研究进展[J].杭州师范大学学报(自然科学版),2015,30(6):608-615.
[15] 罗永明,刘爱华,李琴,等.植物萜类化合物的生物合成途径及其关键酶的研究进展[J].江西中医药大学学报,2003,15(1):46-49.
[16] 陈建,赵德刚.植物萜类生物合成相关酶类及其编码基因的研究进展[J].分子植物育种,2004,2(6):757-764.
[17] 孙丽超,李淑英,王凤忠,等.萜类化合物的合成生物学研究进展[J].生物技术通报,2017(1):64-75.
[18] 顾洋,李江华,堵国成,等.微生物代谢工程的研究进展和展望[J].生物产业技术,2017(1):64-70.
[2] SWAMY M K,SINNIAH U R.A comprehensive review on the phytochemical constituents and pharmacological activities of Pogostemon cablin Benth.:an aromatic medicinal plant of industrial importance [J].Molecules,2015,20(5):8521-8547.
[3] 张仲敏,黎玉翠,彭绍忠,等.广藿香醇研究概述[J].中国中医药信息杂志,2012,19(1):110-112.
[4] DEGUERRY F,PASTORE L,WU S,et al.The diverse sesqui-terpene profile of patchouli,Pogostemon cablin,is correlated with a limited number of sesquiterpene synthases [J].Arch Biochem Biophys,2006,454(2):123-136.
[5] FRISTER T,HARTWIG S,ALEMDAR S,et al.Characterisation of a recombinant patchoulol synthase variant for biocatalytic production of terpenes [J].Appl Biochem Biotechnol,2015,176(8):2185-2201.
[6] WU Shuiqing,SCHALK M,CLARK A,et al.Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants [J].Nat Biotechnol,2006,24(11):1441-1447.
[7] HARTWIG S,FRISTER T,ALEMDAR S,et al.Expression,purification and activity assay of a patchoulol synthase cDNA variant fused to thioredoxin in Escherichia coli [J].Protein Express Purif,2014,97:61-71.
[8] 梁宗锁,方誉民,杨东风.植物萜类化合物生物合成与调控及其代谢工程研究进展[J] .浙江理工大学学报(自然科学版),2017,37(2):255-259.
[9] QUAN Jiayuan,TIAN Jingdong.Circular polymerase extension cloning for high-throughput cloning of complex and combi-natorial DNA libraries [J] .Nat Protoc,2011,6(2):242-251.
[10] QUAN Jiayuan,TIAN Jingdong.Circular polymerase extension cloning of complex gene libraries and pathways [J].PLoS One,2009,4(7):e6441.
[11] CHRISTIANSON D W.Structural biology and chemistry of the terpenoid cyclases [J].Chem Rev,2006,106(8):3412-3442.
[12] 张贵星,袁保梅,许培荣,等.改良的降落PCR与普通PCR结果比较[J].郑州大学学报(医学版),2003,38(3):352-354.
[13] 万兵,闫杰,季明春,等.一种优化的PCR方法——降落PCR扩增目的基因[J].江苏临床医学杂志,2002,6(2):127-129.
[14] 马转转,庞潇卿,谌容,等.萜类化合物生物合成途径中关键酶的研究进展[J].杭州师范大学学报(自然科学版),2015,30(6):608-615.
[15] 罗永明,刘爱华,李琴,等.植物萜类化合物的生物合成途径及其关键酶的研究进展[J].江西中医药大学学报,2003,15(1):46-49.
[16] 陈建,赵德刚.植物萜类生物合成相关酶类及其编码基因的研究进展[J].分子植物育种,2004,2(6):757-764.
[17] 孙丽超,李淑英,王凤忠,等.萜类化合物的合成生物学研究进展[J].生物技术通报,2017(1):64-75.
[18] 顾洋,李江华,堵国成,等.微生物代谢工程的研究进展和展望[J].生物产业技术,2017(1):64-70.