中国红豆杉羟化酶基因TcCYP725A22的亚细胞定位及过表达作用分析
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摘要
抗癌药物紫杉醇生物合成途径中羟化酶的发现是当今研究的热点和难点。文中利用前期分析得到的1个新的中国红豆杉羟化酶基因TcCYP725A22(GenBank登录号:MF448646.1),构建了亚细胞定位载体pCAMIBA1303-TcCYP725A22-EGFP,瞬时侵染洋葱表皮细胞,激光共聚焦显微镜观察结果发现该基因编码蛋白定位在细胞膜。进一步构建了植物表达载体pBI121-TcCYP725A22,经根癌农杆菌AgrobacteriumtumefaciensLBA4404介导转化到中国红豆杉细胞中过表达后,利用荧光定量PCR (qRT-PCR)和液质联用(LC-MS)分析TcCYP725A22过表达对紫杉醇生物合成的影响。结果显示,瞬时转化pBI121-TcCYP725A22的红豆杉细胞中TcCYP725A22基因的转录水平明显高于pBI121空载体对照组。随着TcCYP725A22基因过表达,几个已知的紫杉醇合成关键酶基因的表达量也都有不同程度的上调,且细胞中各紫杉烷的含量普遍提高。这些结果说明羟化酶基因TcCYP725A22极有可能参与紫杉醇的生物合成路径。
The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725 A22(GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725 A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane.Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725 A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725 A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725 A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725 A22 is likely involved in the biosynthetic pathway of taxol.
The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725 A22(GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725 A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane.Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725 A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725 A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725 A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725 A22 is likely involved in the biosynthetic pathway of taxol.
引文
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[25]Han YP,Vimolmangkang S,Soria-Guerra RE,et al.Ectopic expression of apple F3'H genes contributes to anthocyanin accumulation in the arabidopsis tt7 mutant grown under nitrogen stress.Plant Physiol,2010,153(2):806-820.
[26]Zhang TT,Ma JW,Wang LY,et al.Improving artemisinin content of Artemisia annua L.through overexpression of flavanone 3-hydroxylase gene(AaF3H).Curr Biotech,2018,8(1):55-62(in Chinese).张婷婷,马嘉伟,王路尧,等.过量表达黄烷酮3-羟化酶基因(AaF3H)提高青蒿中青蒿素的含量.生物技术进展,2018,8(1):55-62.
[2]Ketchum RE,Wherland L,Croteau RB.Stable transformation and long-term maintenance of transgenic Taxus cell suspension cultures.Plant Cell Rep,2007,26(7):1025-1033.
[3]Julsing MK,Koulman A,Woerdenbag HJ,et al.Combinatorial biosynthesis of medicinal plant secondary metabolites.Biomol Eng,2006,23(6):265-279.
[4]Ajikumar PK,Xiao WH,Tyo KEJ,et al.Isoprenoid pathway optim ization for Taxol precursor overproduction in Escherichia coli.Science,2010,330(6000):70-74.
[5]Zhou K,Qiao KJ,Edgar S,et al.Distributing a metabolic pathway among a microbial consortium enhances production of natural products.Nat Biotech,2015,33(4):377-383.
[6]Biggs BW,Lim CG,Sagliani K,et al.Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli.Proc Natl Acad Sci USA,2016,113(12):3209-3214.
[7]Chen QP,Liao WF,Fu CH,et al.Research progress in hydroxylase in taxol biosynthetic pathway.Chin JBiotech,2016,32(5):554-564(in Chinese).陈清浦,廖卫芳,付春华,等.紫杉醇生物合成途径中羟化酶的研究进展.生物工程学报,2016,32(5):554-564.
[8]Eisenreich W,Menhard B,Lee MS,et al.Multiple oxygenase reactions in the biosynthesis of taxoids.J Am Chem Soc,1998,120(37):9694-9695.
[9]Korte F,Kvesitadze G,Ugrekhelidze D,et al.Organic toxicants and plants.Ecotox Environ Saf,2000,47(1):1-26.
[10]Schuler MA.Plant cytochrome P450 monooxygenases.Crit Rev Plant Sci,1996,15(3):235-284.
[11]Hannemann F,Bichet A,Ewen KM,et al.Cytochrome P450 systems-biological variations of electron transport chains.Biochim Biophys Acta,2007,1770(3):330-344.
[12]Paquette SM,Bak S,Feyereisen R.Intron-exon organization and phylogeny in a large superfamily,the paralogous cytochrome P450 genes of Arabidopsis thaliana.DNA Cell Biol,2000,19(5):307-317.
[13]Croteau R,Ketchum REB,Long RM,et al.Taxol biosynthesis and molecular genetics.Phytochem Rev,2006,5(1):75-97.
[14]Jennewein S,Long RM,Williams RM,et al.Cytochrome P450 taxadiene 5α-hydroxylase,a mechanistically unusual monooxygenase catalyzing the first oxygenation step of taxol biosynthesis.Chem Biol,2004,11(3):379-387.
[15]Schoendorf A,Rithner CD,Williams RM,et al.Molecular cloning of a cytochrome P450 taxane10β-hydroxylase cDNA from Taxus and functional expression in yeast.Proc Natl Acad Sci USA,2001,98(4):1501-1506.
[16]Jennewein S,Rithner CD,Williams RM,et al.Taxol biosynthesis:taxane 13α-hydroxylase is a cytochrome P450-dependent monooxygenase.Proc Natl Acad Sci USA,2001,98(24):13595-13600.
[17]Chau M,Croteau R.Molecular cloning and characterization of a cytochrome P450 taxoid2α-hydroxylase involved in taxol biosynthesis.Arch Biochem Biophys,2004,427(1):48-57.
[18]Chau M,Jennewein S,Walker K,et al.Taxol biosynthesis:molecular cloning and characterization of a cytochrome P450 taxoid 7β-hydroxylase.Chem Biol,2004,11(5):663-672.
[19]Guerra-Bubb J,Croteau R,Williams RM.The early stages of taxol biosynthesis:an interim report on the synthesis and identification of early pathway metabolites.Nat Prod Rep,2012,29(6):683-696.
[20]Liao WF,Zhao SY,Zhang,M,et al.Transcriptome assembly and systematic identification of novel cytochrome P450s in Taxus chinensis.Front Plant Sci,2017,8:1468.
[21]Zhang CH,Mei XG,Liu L,et al.Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis.Biotechnol Lett,2000,22(19):1561-1564.
[22]Lee MH,Jeong JH,Seo JW,et al.Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene.Plant Cell Physiol,2004,45(8):976-984.
[23]Kim OT,Kim SH,Ohyama K,et al.Upregulation of phytosterol and triterpene biosynthesis in Centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate synthase.Plant Cell Rep,2010,29(4):403-411.
[24]Zheng Y,Huang YY,Xian WH,et al.Identification and expression analysis of the glycine max cyp707a gene family in response to drought and salt stresses.Ann Botany,2012,110(3):743-756.
[25]Han YP,Vimolmangkang S,Soria-Guerra RE,et al.Ectopic expression of apple F3'H genes contributes to anthocyanin accumulation in the arabidopsis tt7 mutant grown under nitrogen stress.Plant Physiol,2010,153(2):806-820.
[26]Zhang TT,Ma JW,Wang LY,et al.Improving artemisinin content of Artemisia annua L.through overexpression of flavanone 3-hydroxylase gene(AaF3H).Curr Biotech,2018,8(1):55-62(in Chinese).张婷婷,马嘉伟,王路尧,等.过量表达黄烷酮3-羟化酶基因(AaF3H)提高青蒿中青蒿素的含量.生物技术进展,2018,8(1):55-62.