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Acta Agron Sin ›› 2009, Vol. 35 ›› Issue (5): 855-860.doi: 10.3724/SP.J.1006.2009.00855


Transformation of EPSP Synthetase Gene from Allium macrostemon Bunge into Tobacco and Improvement of Resistance in Transgenic Phants

HUANG Li-Hua1,JIANG Xiang1,LI Bo1,LI Yu-Qiang2,ZHANG Xue-Wen1*   

  1. 1College of Bioscience and Biotechnology,Hunan Agricultural University, Changsha 410128,China;2Huna Cotton Science Institute, Changde 415101,Hunan,China
  • Received:2008-09-03 Revised:2008-12-30 Online:2009-05-12 Published:2009-02-16
  • Contact: ZHANG Xue-Wen E-mail:xwzhang@hunau.net


EPSPs (5-enolppyruvylshikimate-3-phosphate synthetase) is an important enzyme involved in the synthesis of the aromatic amino acids in all plants, which catalyzes phosphoenolpyruvate and shikimate 3-phophate to form 5-enolpyruvlshimimate-3-phosphate and phosphate. Glyphosate is a chemical with similar structure of phosphoenolpyruvate, which combines enzyme to inhibit the activity of EPSPs, so it is a broad spectrum herbicide to control weeds. Breeding glyphosate tolerance or resistance crops will be an effective way to control weeds. In the study, a gene cDNA (EPSPsA) isolated from Allium macrostemon Bunge was ligated with vector pWM101 to construct a recombinant vector. The recombinant gene was transformed into WS38 via Agrobacterium-mediated way to obtain transgenic tobacco seedlings. Transgenic seedlings were screened with ordinary antibiotics. PCR analysis showed that EPSPsA was successfully integrated into the tobacco genome in all the transgenic seedlings. The result of RT-PCR also validated that EPSPsA was transcripted at mRNA leve1. At 60 d after spraying 200 mg L-1 glyphosate, the transgenic tobacco plants grew better than the untransformed plants. The average fresh weight of aerial parts in transgenic plants had a significant increase compared with those in untransformed control plants. The average chlorophyll content in transgenic plants was 5.54 mg g-1, while that in the control only 1.16 mg g-1. In the treatment with 400 mg L-1 glyphosate, the untransformed plants died while the transgenic ones grew well in the treatment with 1 000 mg L-1 glyphosate. These results proved that the EPSPsA can  improve the glyphosate tolerance in tobacco .

Key words: Allium macrostemon Bunge, EPSPs cDNA, Tobacco transformation, Glyphosate resistance

[1] Bentley R. The shikimate pathway—A metabolic tree with many branches. Crit Rev Biochem Mol Biol, 1990, 25: 307–384
[2] Du W S, Wa11is N G, Mazzulla M J, Chalker A F, Zhang L, Liu W S, Kallender H, Payne D J. Characterizaition of Streptococcus pneumoniae 5-enolpynevylshikimate-3-phosphate synthase and its actibation by univalent cations. Eur J Bioche, 2000, 267: 222–227
[3] Baylis A D. Why glyphosate is a global herbicide:strengths, weednesses and prospects. Pest Manag Sci, 2000, 56: 299–308
[4] Ling C-F(林长福), Li Z-N(李志念). Diagnosis preventation and remedy of herbicides phytotoxic effect. Modern Agrochem (现代农药), 2002, (6): 29–31(in Chinese with English abstract)
[5] Duan H-X(段洪晓), Shen M-X(沈明学), Li W(李伟), Lun L-W(伦立武). Reason and counter-measure of weedcide-side occuring. Inner Mongolia Agric Sci Technol (内蒙古农业科技), 2001, (2): 19(in Chinese)
[6] Liang X-L(梁雪莲), Wang Y-B(王引斌), Wei J-Q(卫建强), Gou J-F(缑建芳). The progress of studies on herbicide resistance gene of crop. Biotechnol Bull (生物技术通报), 2001, (2): 17–21(in Chinese with English abstract)
[7] Diao X-M(刁现民). Achievements in herbicide resistant gene engineering of crops. J Hebei Agric Sci (河北农业科学), 2001, 5(1): 70–71(in Chinese with English abstract)
[8] Zhu Y(朱玉), Yu Z-L(于中连), Lin M(林敏). Bioresistance or biodegradation of glyphosate and construction of transgenic Pants. Mol Plant Breed (分子植物育种), 2003, 1(4): 435–441(in Chinese with English abstract)
[9] Sost D, Amrherin N. Substitution of gly-96 to Ala in the 5-enolpyruvylshikimate-3-phosphate synathase of klebsiella pneumoniae results in a grestly reduced affinity for the herbicide glyphosate. Archives Biochem Biophysics, 1990, 282: 433–436
[10] Blackburn L G, Boutin C. Subtle effects of herbicide use in the context of genetically modified crops: A case study with glyphosate. Ecotoxicology, 2003, 12: 271–285
[11] Maskell D. Cloning and sequencing of the haemophilus influenzae aroA gene of Bordetella pertussis. J Bacteriol, 1998, 180: 2467–2471
[12] Gallo M, Irvine J E. Herbicide resistant transgenic sugarcane plants containing the Bar gene. Crop Sci, 1996, 36: 1367–1374
[13] Zhou H, Arrowsmith J W, Fromm M E, Hironaka C M, Taylor M L, Rodriguez D, Pajeau M E, Brown S M, Santino C G, Fry J E. Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation. Plant Cell Rep, 1995, 15: 157–159
[14] Mannerlof M, Tuvesson S, Steen P, Tennung P. Transgenic sugar beet tolerant to glyphosate. Euphytica, 1997, 94: 83–91
[15] Penaloza A, Mena G L, Herrera L, Bailey A M. Cloning and sequencing of the genes involved in glyphosate utilization by Pseudomonas pseudomallei. Appl Environ Microbio, 1995, 61: 538–543
[16] Zboinska E, Lejezak B, Kafrski P. Orgnophosphonate utilization by the wild-type strain of Pseudomonas fluorescens. Appl Environ Microbio, 1992, 58: 2993–2999
[17] Scott R, Damian J, Minhtien T, Feng Y M, Nancy A, Gerald M. Glyphosate-resistant goosegrass, identification of a mutationin the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Plant Physiol, 2002, 129: 1265–1275
[18] Zhou M, Xu H L, Wei X L, Ye Z Q, Wei L P, Gong W M, Wang Y Q, Zhu Z. Identification of a Glyphosate–resistant mutant of rice5- enolpyruvylshikimate 3-phosphate synthase using adirected evolution strategy. Plant Physiol, 2006, 140: 184–195
[19] Jiang X(蒋向), Dai Z-X(戴雄泽), Li Y-Q(李育强), Huang L-H(黄丽华), Dai Q(戴倩), Zhang X-W(张学文). Semi-quantitative analysis of EPSPs gene expression in tissues of Allium macrostemon Bunge. J Hunan Agric Univ (湖南农业大学学报), 2007, 33(5): 542–545(in Chinese with English abstract)
[20] Roberts C W, Robetrs F, Lyons R E, Kirisits M, JMui E J, Finnerty J, Johnson J J, Ferguson D J, Coggins J R, Krell T, Coombs G H, Milhous W K, Kyle D E, Tzipori S, Barnwell J, Dame J B, Carlton J, McLeod R. The shikimate pathway and its branches in apicomplexan parasites. J Infect Dis, 2002, 185: 25–36
[21] Wang H-W(王宏伟), Liang Y-H(梁业红), Shi Z-S(史振声), Zhang S-H(张世煌). Review of study on transgenic crop resistance to glyphosate. Crops (作物杂志), 2007, (4): 9–12(in Chinese)
[22] Rogers S G, Brand L A, Holder S B. Amplification of the aroA gene from Escherichia coli results in tolerance to the gerbicide glyphosate. Appl Environ Microbiol, 1983, 46: 37–43
[23] Shah D M, Hosrch R B, Klee H J, Kishore G M, Winter J A, Tumer N E, Hironak C M, Sanders P R , Gasser C S, Siegel N R, Rogers N G, Fraley R T. Engineeirng herbicide tolerance in transgenic plants. Science, 1986, 233: 478–481
[24] Lebrun M, Sailland A, Freyssinet G. Mutated 5-enolpyruvylshikimate-3-phosphate synthase, gene coding for said protein and transformed plants containing said gene. US Patent, 2003, 6: 566–587
[25] Chen H-L(陈梁鸿), Wang X-W(王新望), Zhang W-J(张文俊), Zhang X-D(张晓东), Hu D-F(胡道芬), Liu G-T(刘广田). Transformation of common wheat with herbicide-resisitant EPSPs gene. Acta Genet Sin (遗传学报), 1999, 26(3): 239–243(in Chinese with English abstract)
[26] Yang H(杨勇), Zhang X-W(张学文). Callus induction of Allium macrostemon Bunge and its resistance to glyphosate. J Hunan Agric Univ (湖南农业大学学报), 2007, 33(3): 277–280(in Chinese with English abstract)
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