雷公藤牻牛儿基焦磷酸合酶基因TwGPPS克隆与表达分析
|
摘要
根据雷公藤悬浮细胞转录组数据设计特异引物,采用cDNA末端快速扩增技术克隆雷公藤牻牛儿基焦磷酸合酶基因TwGPPS1,TwGPPS2全长cDNA,并进行生物信息学分析和蛋白表达研究。首次克隆得到TwGPPS1核苷酸的完整开放阅读框长度为1 278 bp,编码425个氨基酸蛋白,预测蛋白等电点为6.68,相对分子质量为47.189 kDa;TwGPPS2核苷酸的完整开放阅读框长度为1 269 bp,编码422个氨基酸蛋白,预测蛋白等电点为6.71,相对分子质量为46.774 kDa;进一步构建了原核重组表达载体pET-32a-TwGPPS1,pET-32a-TwGPPS2,并成功诱导出TwGPPS1,TwGPPS2重组蛋白,为深入研究此关键酶基因功能和雷公藤萜类生物合成途径奠定基础。
Based on the transcriptome data,the study cloned full-length c DNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a1 278 bp open reading frame( ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point( p I) was 6. 68,a calculated molecular weight was about 47. 189 k Da. The full-length c DNA of the TwGPPS2 contains a 1 269 bp open reading frame(ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point(p I) was 6. 71,a calculated molecular weight was about 46. 774 k Da. The entire reading frame of TwGPPS1,2 was cloned into the p ET-32 a( +) vector and expressed in E. coli BL21( DE3) cells to obtain the TwGPPS protein,which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.
Based on the transcriptome data,the study cloned full-length c DNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a1 278 bp open reading frame( ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point( p I) was 6. 68,a calculated molecular weight was about 47. 189 k Da. The full-length c DNA of the TwGPPS2 contains a 1 269 bp open reading frame(ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point(p I) was 6. 71,a calculated molecular weight was about 46. 774 k Da. The entire reading frame of TwGPPS1,2 was cloned into the p ET-32 a( +) vector and expressed in E. coli BL21( DE3) cells to obtain the TwGPPS protein,which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.
引文
[1]刘为萍,刘素香,唐慧珠,等.雷公藤研究新进展[J].中草药,2010,41(7):1215.
[2]林光美,张敏,侯长红.雷公藤研究进展[J].中国农学通报,2009,25(23):90.
[3]张秋萍,宋洪涛.雷公藤制剂研究进展[J].中国药师,2009,12(7):881.
[4]黄明来,马卓.雷公藤的研究进展[J].化学与生物工程,2012,29(7):1.
[5]斯金平,阮秀春,郭宝林,等.雷公藤资源现状及可持续利用的研究[J].中药材,2005,28(1):10.
[6]王彩云,李富生,李涛,等.滇龙胆Gr GPPS基因的克隆及其序列分析与原核表达[J].中草药,2014,45(15):2060.
[7]于盱,王海棠,冯美英,等.薄荷GPPS基因的克隆与表达分析[N].江西农业学报,2013,25(7):25.
[8]于盱,梁呈元,刘艳,等.薄荷GPPS基因原核表达及RNA干扰载体构建[J].生物技术通报,2014(9):84.
[9]Rai A,Smita S S,Singh A K,et al.Heteromeric and homomeric geranyl diphsophate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis[J].Mol Plant,2013,6(5):1531.
[10]Dorothea Tholl,Christine M Kish,Irina Orlova,et al.Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases[J].Plant Cell,2004,16(4):977.
[11]Burke C,Croteau R.Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate[J].J Biol Chem,2002,277(5):3141.
[12]Chang T H,Hsieh F L,Ko T P,et al.Structure of a heterotetrameric geranyl pyrophosphate synthase from mint(Mentha piperita)reveals intersubunit regulation[J].Plant Cell,2010,22(2):454.
[13]Wang Guodong,Richard A Dixon.Heterodimeric geranyl(geranyl)diphosphate synthase from hop(Humulus lupulus)and the evolution of monoterpene biosynthesis[J].Proc Natl Acad Sci USA,2009,106(24):9914.
[2]林光美,张敏,侯长红.雷公藤研究进展[J].中国农学通报,2009,25(23):90.
[3]张秋萍,宋洪涛.雷公藤制剂研究进展[J].中国药师,2009,12(7):881.
[4]黄明来,马卓.雷公藤的研究进展[J].化学与生物工程,2012,29(7):1.
[5]斯金平,阮秀春,郭宝林,等.雷公藤资源现状及可持续利用的研究[J].中药材,2005,28(1):10.
[6]王彩云,李富生,李涛,等.滇龙胆Gr GPPS基因的克隆及其序列分析与原核表达[J].中草药,2014,45(15):2060.
[7]于盱,王海棠,冯美英,等.薄荷GPPS基因的克隆与表达分析[N].江西农业学报,2013,25(7):25.
[8]于盱,梁呈元,刘艳,等.薄荷GPPS基因原核表达及RNA干扰载体构建[J].生物技术通报,2014(9):84.
[9]Rai A,Smita S S,Singh A K,et al.Heteromeric and homomeric geranyl diphsophate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis[J].Mol Plant,2013,6(5):1531.
[10]Dorothea Tholl,Christine M Kish,Irina Orlova,et al.Formation of monoterpenes in Antirrhinum majus and Clarkia breweri flowers involves heterodimeric geranyl diphosphate synthases[J].Plant Cell,2004,16(4):977.
[11]Burke C,Croteau R.Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate[J].J Biol Chem,2002,277(5):3141.
[12]Chang T H,Hsieh F L,Ko T P,et al.Structure of a heterotetrameric geranyl pyrophosphate synthase from mint(Mentha piperita)reveals intersubunit regulation[J].Plant Cell,2010,22(2):454.
[13]Wang Guodong,Richard A Dixon.Heterodimeric geranyl(geranyl)diphosphate synthase from hop(Humulus lupulus)and the evolution of monoterpene biosynthesis[J].Proc Natl Acad Sci USA,2009,106(24):9914.