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Overexpression of a modified AM79 aroA gene in transgenic maize confers high tolerance to glyphosate |
REN Zhen-jing, CAO Gao-yi, ZHANG Yu-wen, LIU Yan, LIU Yun-jun |
1、Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
2、College of Agriculture and Biotechnology, China Agricultural University, Beijing 100094, P.R.China |
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摘要 It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops (Cao et al. 2012). In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cells. The redesigned and artificially synthesized gene, named as mAM79, was cloned into plant expression vector pM3301UbiSpAM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase (rbcS) small subunit and controlled by ubiquitin promoter. The plasmid was transformed into maize (Zea mays) immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate; however, all the non-transgenic maize plants were killed by glyphosate. The results in this study confirmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.
Abstract It has previously been shown that a bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene AM79 aroA can be a candidate gene to develop glyphosate-tolerant transgenic crops (Cao et al. 2012). In this study, AM79 aroA was redesigned using the plant biased codons and eliminating the motifs which would lead to the instability of mRNA, to create a synthetic gene that would be expressed highly in plant cells. The redesigned and artificially synthesized gene, named as mAM79, was cloned into plant expression vector pM3301UbiSpAM79, where mAM79 is fused with signal peptide sequence of pea rib-1,5-bisphospate carboxylase (rbcS) small subunit and controlled by ubiquitin promoter. The plasmid was transformed into maize (Zea mays) immature embryos using Agrobacterium-mediated transformation method. Total 74 regenerated plants were obtained and PCR analysis showed that these transgenic plants had the integration of mAM79. Southern blot analysis was performed on the genomic DNA from four transgenic lines, and the result showed that one or two copies of mAM79 were integrated into maize genome. RT-PCR analysis result indicated that mAM79 was highly transcribed in transgenic maize plants. When sprayed with glyphosate, transgenic maize line AM85 and AM72 could tolerate 4-fold of commercial usage of glyphosate; however, all the non-transgenic maize plants were killed by glyphosate. The results in this study confirmed that mAM79 could be used to develop glyphosate-tolerant maize, and the obtained transgenic maize lines could be used for the breeding of glyphosate-tolerant maize.
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Received: 29 August 2014
Accepted:
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Fund: This work was funded by the National Major Project for Transgenic Organism Breeding, China (2014ZX08010-004). |
Corresponding Authors:
LIU Yun-jun, Tel/Fax: +86-10-82105863,E-mail: liuyunjun@caas.cn
E-mail: liuyunjun@caas.cn
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Cite this article:
REN Zhen-jing, CAO Gao-yi, ZHANG Yu-wen, LIU Yan, LIU Yun-jun.
2015.
Overexpression of a modified AM79 aroA gene in transgenic maize confers high tolerance to glyphosate. Journal of Integrative Agriculture, 14(3): 414-422.
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Belfiore N M, Anderson S L. 2001. Effects of contaminants ongenetic patterns in aquatic organisms: A review. MutantResearch, 489, 97-122Bradshaw L, Padgette S, Kimball S, Wells B. 1997. Perspectiveson glyphosate resistance. Weed Technology, 11, 189-198Cao G, Liu Y, Zhang S, Yang X, Chen R, Zhang Y, Lu W, WangJ, Lin M, Wang G. 2012. A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance inEscherichia coli and tobacco plants. PLoS ONE, 7, e38718.Dennehey B, Petersen W, Ford-Santino C, Pajeau M, ArmstrongC. 1994. Comparison of selective agents for use with theselectable marker gene bar in maize transformation. PlantCell Tissue Organ Culture, 36, 1-7Dun B Q, Wang X J, Lu W, Zhao Z L, Hou S N, Zhang B M, LiG Y, Evans Jr T C, Xu M Q, Lin M. 2007. Reconstitutionof glyphosate resistance from a split 5-enolpyruvylshikimate-3-phosphate synthase gene in Escherichiacoli and transgenic tobacco. Applied and EnvironmentalMicrobiology, 73, 7997-8000Finkelstein R, Lynch T, Reeves W, Petitfils M, MostachettiM. 2011. Accumulation of the transcription factor ABAinsensitive(ABI)4 is tightly regulated post-transcriptionally.Journal of Experimental Botany, 62, 3971-3979Foster P L. 2000. Adaptive mutation: Implications for evolution.BioEssays, 22, 1067-1074Franz J E, Mao M K, Sikorski J A. 1997. Glyphosate: A uniqueglobal herbicide. American Chemical Society Monograph,189, 521-615Funke T, Han H, Healy-Fried M L, Fischer M, SchonbrunnE. 2006. Molecular basis for the herbicide resistanceof Roundup Ready crops. Proceedings of the NationalAcademy of Sciences of the United States of America,103, 13010-13015Funke T, Yang Y, Han H, Healy-Fried M, Olesen S, BeckerA, Schonbrunn E. 2009. Structural basis of glyphosateresistance resulting from the double mutation Thr97 → Ileand Pro101 → Ser in 5-enolpyruvylshikimate-3-phosphatesynthase from Escherichia coli. The Journal of BiologicalChemistry, 284, 9854-9860Goodall G J, Filipowicz W. 1989. The AU-rich sequencespresent in the introns of plant nuclear pre-mRNAs arerequired for splicing. Cell, 58, 473-483He M, Yang Z Y, Nie Y F, Wang J, Xu P. 2001. A new typeof class I bacterial 5-enopyruvylshikimate-3-phosphatesynthase mutants with enhanced tolerance to glyphosate.Biochimica et Biophysica Acta, 1568, 1-6Herrmann K M, Weaver L M. 1999. The shikimate pathway.Annual Review Plant Physiology and Plant MolecularBiology, 50, 473-503Howe A, Gasser C, Brown S, Padgette S, Hart J, Parker G,Fromm M, Armstrong C. 2002. Glyphosate as a selectiveagent for the production of fertile transgenic maize (Zeamays) plants. Molecular Breeding, 10, 153-164Hu T, Metz S, Chay C, Zhou H P, Biest N, Chen G, Cheng M,Feng X, Radionenko M, Lu F, Fry J. 2003. Agrobacteriummediatedlarge-scale transformation of wheat (Triticumaestivum L.) using glyphosate selection. Plant Cell Reports,21, 1010-1019Jin D, Lu W, Ping S, Zhang W, Chen J, Dun B, Ma R, Zhao Z,Sha J, Li L, Yang Z, Chen M, Lin M. 2007. Identification of anew gene encoding EPSPs with high glyphosate resistancefrom the metagenomic library. Current Microbiology, 55,350-355Joshi C P. 1987. Putative polyadenylation signals in nucleargenes of higher plants: A compilation and analysis. NucleicAcids Research, 15, 9627-9640Koziel M G, Beland G L, Bowman C, Carozzi N B, Crenshaw R,Crossland L, Dawson J, Desai N, Hill M, Kadwell S, LaunisK, Lewis K, Maddox D, McPherson K, Meghji M R, Merlin E,Rhodes R, Warren G W, Wright M, Evola S V. 1993. Fieldperformance of elite transgenic maize plants expressingan insecticidal protein derived from Bacillus thuringiensis.Nature Biotechnology, 11, 194-200Liang A, Sha J, Lu W, Chen M, Li L, Jin D, Yan Y, Wang J, PingS, Zhang W, Wang Y, Lin M. 2008. A single residue mutationof 5-enoylpyruvylshikimate-3-phosphate synthase inPseudomonas stutzeri enhances resistance to the herbicideglyphosate. Biotechnology Letters, 30, 1397-1401Lin M, Liang A, Lu W, Li L, Chen M, Zhang W, Ping S. 2007.An EPSPS synthase with high glyphosate resistance andencoded sequence. World Patent, WO/2009/059485.Liu D. 2009. Design of gene constructs for transgenic maize.Methods in Molecular Biology, 526, 3-20Liu Y, Zhang Y, Liu Y, Lu W, Wang G. 2014. Metabolic effectsof glyphosate on transgenic maize expressing a G2-EPSPS gene from Pseudomonas fluorescens. Journal of PlantBiochemistry and Biotechnology, doi: 10.1007/s13562-13014-10263-13569.Liu Z, Pan Z, Xu Y, Dong Z, Yang Z, Lin M. 2006. Cloningand expression of a 5-enolpyruvylshikimate-3-phosphatesynthase gene from Halomonas variabilis. DNA Sequence,17, 208-214Lopez-Rodas V, Flores-Moya A, Maneiro E, Perdigones N,Marva F, Garc?a M E, Costas E. 2007. Resistance toglyphosate in the cyanobacterium Microcystis aeruginosaas result of pre-selective mutations. Evolutionary Ecology,21, 535-547Murray M, Thompson W F. 1980. Rapid isolation of highmolecular weight plant DNA. Nucleic Acids Research, 8,4321-4326Shou H, Frame B, Whitham S, Wang K. 2004. Assessment oftransgenic maize events produced by particle bombardmentor Agrobacterium-mediated transformation. MolecularBreeding, 13, 201-208Steinrucken H C, Amrhein N. 1980. The herbicide glyphosate isa potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphatesynthase. Biochemical and Biophysical ResearchCommunications, 94, 1207-1212Vaucheret H, Beclin C, Elmayan T, Feuerbach F, Godon C,Morel J, Mourrain P, Palauqui J, Vernhetters S. 1998.Transgene induced gene silencing in plants. The PlantJournal, 16, 651-659Walters D A, Vetsch C S, Potts D E, Lundquist R C.1992. Transformation and inheritance of a hygromycinphosphotransferase gene in maize plants. Plant MolecularBiology, 18, 189-200Wang H Y, Li Y F, Xie L X, Xu P. 2003. Expression of a bacterialaroA mutant, aroA-M1, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosateresistanttobacco plants. Journal of Plant Research, 116,455-460Ye G N, Hajdukiewicz P T, Broyles D, Rodriguez D, XuC W, Nehra N, Staub J M. 2001. Plastid-expressed5-enolpyruvylshikimate-3-phosphate synthase genesprovide high level glyphosate tolerance in tobacco. ThePlant Journal, 25, 261-270Zhang S, Lian Y, Liu Y, Wang X, Liu Y, Wang G. 2013.Characterization of a maize Wip1 promoter in transgenicplants. International Journal of Molecular Sciences, 14,23872-23892Zhou M, Xu H, Wei X, Ye Z, Wei L, Gong W, Wang Y, ZhuZ. 2006. Identification of a glyphosate-resistant mutant ofrice 5-enolpyruvylshikimate 3-phosphate synthase using adirected evolution strategy. Plant Physiology, 140, 184-195 |
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