基于核糖体跳跃的多顺反子共表达技术在拟南芥上的应用
|
摘要
2A肽的核糖体跳跃技术是一种新型技术。其2A肽是长度为18～22个氨基酸的顺式作用水解酶元件,具有结构短小、蛋白分割效率高的特点,因而被越来越多应用于多顺反子表达载体的构建中。该项技术目前主要集中于酵母(Saccharomyces cerevisiae)和哺乳动物细胞的研究中,在植物中的报道较少。为探索该技术在植物中的应用研究,本研究利用猪捷申病毒2A (Porcine teschovirus-1 2A, P2A)和东亚细亚病毒2A (Thosea asigna virus 2A, T2A),首次将人工合成的转录因子XVE (X, the DNA-binding domain of the bacterial repressor LexA; V, the acidic transactivating domain of VP16; E, the regulatory region of the human estrogen receptor)、绿色荧光蛋白基因(green fluorescent protein, GFP)和抗除草剂基因(bialaphos resistance,Bar)3个基因的开放阅读框(open reading fragment, ORF)串联后受35S启动子调控,构建成多顺反子共表达载体pX6-2A,转化拟南芥(Arabidopsis thaliana)后经PCR鉴定获得了转基因阳性植株;Western blot结果显示XVE,GFP和Bar 3个目的蛋白全部被分割完成,说明P2A和T2A肽在植物材料中可以转录、表达和翻译,且在分割过程中不会互相干扰,是一种理想的多顺反子表达方式。本研究结果表明P2A和T2A在植物材料中能够完成高效分割,行使其"核糖体跳跃"的功能,适用于在植物材料中的应用研究,该研究结果对2A肽在转基因植物中的推广应用具有重要的理论和借鉴意义。
2A peptide is a novel ribosomal skipping based technique, which takes advantage of a cis-acting hydrolase element of a length of 18～22 amino acids with profiles of short structure and high protein segmentation efficiency. Therefore, it is increasingly used in polycistronic expression vectors. At present, it is widely used in yeast(Saccharomyces cerevisiae) and mammal cells, less in plants. In order to explore 2A peptide application in plants, the polycistronic co-expression vector pX6-2A with Porcine teschovirus-1 2A(P2A) and Thosea asigna virus 2A(T2A) linking 3 ORFs(open reading fragments) including a chimeric transcription activator gene XVE(X, the DNA-binding domain of the bacterial repressor LexA; V, the acidic trans-activating domain of VP16; E, the regulatory region of the human estrogen receptor.), GFP(green fluorescent protein) and Bar(bialaphos resistance) in series under the control of 35 S promoter was constructed, and transformed into Arabidopsis thaliana, to obtain successful transgenic plants. Western blot results showed that these 3 proteins XVE, GFP and Bar expressed separately, indicated that both P2A and T2A peptide could transcribe, express and translate normally, and there was no interference with each other. It suggested that it could be an ideal way to express polycistronic in plants. These results indicated that P2A and T2A could do 'ribosome skipping' and separate proteins completely in plants. P2A and T2A were suitable for application in plants, which provides an important theoretical and referential significance for the popularization and application of 2A peptide in transgenic plants.
2A peptide is a novel ribosomal skipping based technique, which takes advantage of a cis-acting hydrolase element of a length of 18～22 amino acids with profiles of short structure and high protein segmentation efficiency. Therefore, it is increasingly used in polycistronic expression vectors. At present, it is widely used in yeast(Saccharomyces cerevisiae) and mammal cells, less in plants. In order to explore 2A peptide application in plants, the polycistronic co-expression vector pX6-2A with Porcine teschovirus-1 2A(P2A) and Thosea asigna virus 2A(T2A) linking 3 ORFs(open reading fragments) including a chimeric transcription activator gene XVE(X, the DNA-binding domain of the bacterial repressor LexA; V, the acidic trans-activating domain of VP16; E, the regulatory region of the human estrogen receptor.), GFP(green fluorescent protein) and Bar(bialaphos resistance) in series under the control of 35 S promoter was constructed, and transformed into Arabidopsis thaliana, to obtain successful transgenic plants. Western blot results showed that these 3 proteins XVE, GFP and Bar expressed separately, indicated that both P2A and T2A peptide could transcribe, express and translate normally, and there was no interference with each other. It suggested that it could be an ideal way to express polycistronic in plants. These results indicated that P2A and T2A could do 'ribosome skipping' and separate proteins completely in plants. P2A and T2A were suitable for application in plants, which provides an important theoretical and referential significance for the popularization and application of 2A peptide in transgenic plants.
引文
黄惠,胡广东,康健,等.2014.一种通用型piggyBac转座子诱导细胞永生化载体的构建及其基本功能验证[J].生物工程学报,30(8):1182-1192.(Huang H,Hu G D,Kang J,et al.2014.Construction of a general piggyBac transposon inducible cell immortalization vector and verification of its basic properties[J].Chinese Journal of Biotechnology,30(8):1182-1192.)
吴立柱,王省芬,李喜焕,等.2014.通用型植物表达载体pCamE的构建及功能验证[J].农业生物技术学报,22(6):661-671.(Wu L Z,Wang X F,Li X H,et al.Construction and function identitication of universal plant expression vector pCamE[J].Journal of Agriculural Biotechnology,22(6):661-671.)
Burén S,Ortega-Villasante C,Otv?s K,et al.2012.Use of Foot-and-Mouth disease virus 2A peptide co-expression system to study intracellular protein trafficking in Arabidopsis[J].PLoS One,7(12):e51973.
Chng J,Wang T,Nian R,et al.2015.Cleavage efficient 2Apeptides for high level monoclonal antibody expression in CHO cells[J].MAbs,2015,7(2):403-412.
Clough S J,Bent A F.1998.Floral dip:A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J].The Plant Journal,16(6):735-743.
Donnelly M L,Luke G,Mehrotra A,et al.2001.Analysis of the aphthovirus 2A/2B polyprotein'cleavage'mechanism indicates not a proteolytic reaction,but a novel translational effect:A putative ribosomal'skip'[J].Journal of General Virology,82(5):1013-1025.
Doronina V A,Wu C,Felipe P D,et al.2008.Site-specific release of nascent chains from ribosomes at a sense codon[J].Molecular and Cellular Biology,28(13):4227-4239.
Felipe P D,Ryan M D.2004.Targeting of proteins derived from self-processing polyproteins containing multiple signal sequences[J].Traffic,5(8):616-626.
Felipe P D.2002.Polycistronic viral vectors[J].Current Gene Therapy,2(3):355-378.
Jiao X,Zhang Q,Zhang S,et al.2018.Efficient co-expression of multiple enzymes from a single promoter mediated by virus 2A sequence in the oleaginous yeast rhodosporidium toruloides[J].FEMS Yeast Research,18(8).
Kim J H,Lee S R,Li L H,et al.2011.High cleavage efficiency of a 2A peptide derived from Porcine teschovirus-1 in human cell lines,zebrafish and mice[J].PLoS One,6(4):e18556.
Kuzmich A I,Vvedenski?A V,Kopantsev E P,et al.2013.Quantitative comparison of expression for genes linked in bicistronic vectors via ires or 2A-peptide of Porcine teschovirus-1 sequence[J].Bioorg Khim,39(4):454-465.
Liu Z,Chen O,Wall J B J,et al.2017.Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector[J].Scientific Reports,7(1):2193.
Lorens J B,Pearsall D M,Swift S E,et al.2004.Stable,stoichiometric delivery of diverse protein functions[J].Journal of Biochemical and Biophysical Methods,58(2):101-110.
Luke G A,Ryan M D.2018.Using the 2A protein coexpressionsystem:Multicistronic 2A vectors expressing gene(s)of interest and reporter proteins[J].Methods in Molecular Biology,1755:31-48.
Lynch S E,Gilkerson J R,Symes S J,et al.2011.Equine rhinitis a virus-like particle expressing DNA vaccine induces a virus neutralising immune response in mice[J].Virus Research,158(1-2):294-297.
Momose F,Morikawa Y.2016.Polycistronic expression of the influenza a virus RNA-dependent RNA polymerase by using the thosea asigna virus 2A-like self-processing sequence[J].Frontiers in Microbiology,7:288.
Palmenberg A C,Parks G D,Hall D J,et al.1992.Proteolytic processing of the cardioviral P2 region:Primary 2A/2Bcleavage in clone-derived precursors[J].Virology,190(2):754-762.
R?der J,Fischer R,Commandeur U.2017.Adoption of the2A ribosomal skip principle to Tobacco mosaic virus for Peptide Display[J].Frontiers in Plant Science,8:1125.
Ryan M D,Drew J.1994.Foot-and-mouth disease virus 2Aoligopeptide mediated cleavage of an artificial polyprotein[J].The EMBO Journal,13(4):928-933.
Souza-Moreira T M,Navarrete C,Chen X,et al.2018.Screening of 2A peptides for polycistronic gene expression in yeast[J].FEMS Yeast Research,18(5):foy036.
Sun H,Zhou N,Wang H,et al.2017.Processing and targeting of proteins derived from polyprotein with 2A and LP4/2A as peptide linkers in a maize expression system[J].Plos One,12(3):e0174804.
Szymczak A L,Workman C J,Wang Y,et al.2004.Correction of multi-gene deficiency in vivo using a single"selfcleaving"2A peptide-based retroviral vector[J].Nature Biotechnology,22(5):589-594.
Szymczak-Workman A L,Vignali K M,Vignali D A.2012.Design and construction of 2A peptide-linked multicistronic vectors[J].Cold Spring Harbor Protocols,6(2):199-204.
Yan J,Wang H,Xu Q,et al.2010.Signal sequence is still required in genes downstream of"autocleaving"2A peptide for secretary or membrane-anchored expression[J].Analytical Biochemistry,399(1):144-146.
Zhang B,Rapolu M,Kumar S,et al.2017.Coordinated protein co-expression in plants by harnessing the synergy between an intein and a viral 2A peptide[J].Plant Biotechnology Journal,15:718-728.
吴立柱,王省芬,李喜焕,等.2014.通用型植物表达载体pCamE的构建及功能验证[J].农业生物技术学报,22(6):661-671.(Wu L Z,Wang X F,Li X H,et al.Construction and function identitication of universal plant expression vector pCamE[J].Journal of Agriculural Biotechnology,22(6):661-671.)
Burén S,Ortega-Villasante C,Otv?s K,et al.2012.Use of Foot-and-Mouth disease virus 2A peptide co-expression system to study intracellular protein trafficking in Arabidopsis[J].PLoS One,7(12):e51973.
Chng J,Wang T,Nian R,et al.2015.Cleavage efficient 2Apeptides for high level monoclonal antibody expression in CHO cells[J].MAbs,2015,7(2):403-412.
Clough S J,Bent A F.1998.Floral dip:A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J].The Plant Journal,16(6):735-743.
Donnelly M L,Luke G,Mehrotra A,et al.2001.Analysis of the aphthovirus 2A/2B polyprotein'cleavage'mechanism indicates not a proteolytic reaction,but a novel translational effect:A putative ribosomal'skip'[J].Journal of General Virology,82(5):1013-1025.
Doronina V A,Wu C,Felipe P D,et al.2008.Site-specific release of nascent chains from ribosomes at a sense codon[J].Molecular and Cellular Biology,28(13):4227-4239.
Felipe P D,Ryan M D.2004.Targeting of proteins derived from self-processing polyproteins containing multiple signal sequences[J].Traffic,5(8):616-626.
Felipe P D.2002.Polycistronic viral vectors[J].Current Gene Therapy,2(3):355-378.
Jiao X,Zhang Q,Zhang S,et al.2018.Efficient co-expression of multiple enzymes from a single promoter mediated by virus 2A sequence in the oleaginous yeast rhodosporidium toruloides[J].FEMS Yeast Research,18(8).
Kim J H,Lee S R,Li L H,et al.2011.High cleavage efficiency of a 2A peptide derived from Porcine teschovirus-1 in human cell lines,zebrafish and mice[J].PLoS One,6(4):e18556.
Kuzmich A I,Vvedenski?A V,Kopantsev E P,et al.2013.Quantitative comparison of expression for genes linked in bicistronic vectors via ires or 2A-peptide of Porcine teschovirus-1 sequence[J].Bioorg Khim,39(4):454-465.
Liu Z,Chen O,Wall J B J,et al.2017.Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector[J].Scientific Reports,7(1):2193.
Lorens J B,Pearsall D M,Swift S E,et al.2004.Stable,stoichiometric delivery of diverse protein functions[J].Journal of Biochemical and Biophysical Methods,58(2):101-110.
Luke G A,Ryan M D.2018.Using the 2A protein coexpressionsystem:Multicistronic 2A vectors expressing gene(s)of interest and reporter proteins[J].Methods in Molecular Biology,1755:31-48.
Lynch S E,Gilkerson J R,Symes S J,et al.2011.Equine rhinitis a virus-like particle expressing DNA vaccine induces a virus neutralising immune response in mice[J].Virus Research,158(1-2):294-297.
Momose F,Morikawa Y.2016.Polycistronic expression of the influenza a virus RNA-dependent RNA polymerase by using the thosea asigna virus 2A-like self-processing sequence[J].Frontiers in Microbiology,7:288.
Palmenberg A C,Parks G D,Hall D J,et al.1992.Proteolytic processing of the cardioviral P2 region:Primary 2A/2Bcleavage in clone-derived precursors[J].Virology,190(2):754-762.
R?der J,Fischer R,Commandeur U.2017.Adoption of the2A ribosomal skip principle to Tobacco mosaic virus for Peptide Display[J].Frontiers in Plant Science,8:1125.
Ryan M D,Drew J.1994.Foot-and-mouth disease virus 2Aoligopeptide mediated cleavage of an artificial polyprotein[J].The EMBO Journal,13(4):928-933.
Souza-Moreira T M,Navarrete C,Chen X,et al.2018.Screening of 2A peptides for polycistronic gene expression in yeast[J].FEMS Yeast Research,18(5):foy036.
Sun H,Zhou N,Wang H,et al.2017.Processing and targeting of proteins derived from polyprotein with 2A and LP4/2A as peptide linkers in a maize expression system[J].Plos One,12(3):e0174804.
Szymczak A L,Workman C J,Wang Y,et al.2004.Correction of multi-gene deficiency in vivo using a single"selfcleaving"2A peptide-based retroviral vector[J].Nature Biotechnology,22(5):589-594.
Szymczak-Workman A L,Vignali K M,Vignali D A.2012.Design and construction of 2A peptide-linked multicistronic vectors[J].Cold Spring Harbor Protocols,6(2):199-204.
Yan J,Wang H,Xu Q,et al.2010.Signal sequence is still required in genes downstream of"autocleaving"2A peptide for secretary or membrane-anchored expression[J].Analytical Biochemistry,399(1):144-146.
Zhang B,Rapolu M,Kumar S,et al.2017.Coordinated protein co-expression in plants by harnessing the synergy between an intein and a viral 2A peptide[J].Plant Biotechnology Journal,15:718-728.