Optimization of Epsps Gene and Development of Double Herbicide Tolerant Transgenic PGMS Rice

Authors
L. H. Deng
L. S. Weng
G. Y. Xiao
Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, 410125, People's Republic of China.
Abstract
The alternative use of non-selective herbicides in different years will reduce the possibility of weeds resistance to herbicide by avoiding the crops constant exposure to herbicide selection pressure. The photoperiod-sensitive genic male sterile (PGMS) rice with herbicide resistance will be convenient to the mechanization of hybrid seed production and weed control. In order to develop double herbicide tolerant transgenic PGMS rice, the optimized Epspsgene and the Bar gene were jointly introduced into 7001S, a japonica PGMS rice, by Agrobacterium-mediated transformation. The coding sequence of Epsps gene was optimized based on the codon usage of rice genes and the function of optimized Epspsgene was validated by real-time PCR analysis, enzyme-linked immunosorbent assay of EPSPS protein, and herbicide resistant assay in the obtained independent transformants. The highest relative expression level of EPSPS protein in leaf reached 9.02% of the total soluble proteins. The transformants of T2 generation withstood at least 3.332 g glyphosate /m2 at seedling stage, which was 2.7 times higher than that reported by Monsanto Company. The transformants harboring the optimized Epsps gene and Bar gene were also resistant to glyphosate and glufosinate simultaneously at seedling stage; the resistant dosages were 0.375 g glufosinate m-2 and 0.833 g glyphosate m-2.

Keywords

1. Barry, G. F., Kishore, G. M., Padgette, S. R. and Stallings, W. C. 1997. Glyphosate-tolerant 5-enolpyruvylshikimate-3- phosphate Synthases. US Patent, 5: 804-425.
2. Della, C. G., Bauer, S. C., Klein, B. K., Shah, D. M., Fraley, R. T. and Kishore, G. M. 1986. Translocation of the Precursor of 5-enolpyruvylshikimate-3-phosphate Synthase in Chloroplasts of Higher Plants in Vitro. Proc. Natl. Acad. Sci., 83: 6873-6877.
3. Deng, C. Y., Song, G. S., Xu, J. W. and Zhu, Z. 2003. Increasing Accumulation Level of Foreign Protein in Transgenic Plants through Protein Targeting. Acta. Botanica. Sinica., 45(9): 1084-1089.
4. Dill, G. M., CaJacob, C. A. and Padgette, S. R. 2008. Glyphosate-resistant Crops: Adoption, Use and Future Considerations. Pest Manag. Sci., 64: 326-331.
5. Duke, S. O. 2011. Glyphosate Degradation in Glyphosate-resistant and -susceptible Crops and Weeds. J. Agric. Food Chem., 59(11): 5835-5841.
6. Fischer, A. J., Areh, C. M., Bayer, D. E. and Hill, J. E. 2000. Herbicide-resistant Echinochloa Oryzoides and E. phyllopogon in California Oryza Sativa Fields. Weed Sci., 48: 225-230.
7. Fitzgibbon, J. E. and Braymer, H. D. 1990. Cloning of A Gene from Pseudomonas Sp. Strain PG2982 Conferring Increased Glyphosate Resistance. Appl. Environ. Microbiol., 56: 3382-3388.
8. Fu, Y. P., Zhu, Z. G., Xiao, H., Hu, G. C., Si, H. M., Yu, Y. H. and Sun, Z. X. 2001. Primary Study on Mechanization of Seed Production of Hybrid Rice by Inducing Bar Gene to Pei’ai 64S. Chin. J. Rice Sci., 15(2): 97-100.
9. Heap, I. 2012. International Survey of Herbicide Resistant Weeds. http://www.weedscience.org/In.asp
10. James, C. 2010. Global Status of Commercialized Biotech/GM Crops: 2010. ISAAA Brief. No.42, http://www.isaaa.org/resources/publications/briefs/42/executivesummary/pdf/Brief%2042%20-%20Executive%20Summary%20-%20English.pdf.
11. James, C. 2011. Global Status of Commercialized Biotech/GM Crops: 2011. ISAAA Brief, No.43, http://www.isaaa.org/resources/publications/briefs/43/executivesummary/pdf/Brief%2043%20-%20Executive%20Summary%20-%20English.pdf
12. Kim, E. H., Suh, S. C., Park, B. S., Shin, K. S., Kweon, S. J., Han, E. J., Park, S. H., Kim, Y. S. and Kim, J. K. 2009. Chloroplast-targeted Expression of Synthetic Cry1Ac in Transgenic Rice as An Alternative Strategy for Increased Pest Protection. Planta, 230: 397-405.
13. Kim, S. S., Jung, J. Y., Jeong, S. K., Lee, D. S., Chen, L. J. and Suh, H. S. 2007. Use of Herbicide-resistant Genic Male Sterility in Hybrid Rice Seed Production. Euphytica, 156: 297-303.
14. Kishore, G. M., Padgette, S. R. and Fraley, R. T. 1992. History of Herbicide Tolerant Crops, Methods of Development and Current State of the Art-emphasis on Glyphosate Tolerance. Weed Techn., 6: 626-634.
15. Kuk, Y. I., Burgos, N. R. and Shiwain, V. K. 2008. Natural Tolerance to Imazethapyr in Red Rice (Oryza sativa). Weed Sci., 56: 1-11.
16. Livak, K. J. and Schmittgen, T. D. 2001. Analysis of Relative Gene Expression Data Using Real-time Quantitative PCR and the 2(-Delta Delta C (T)) Method. Methods, 25: 402-408.
17. Miki, B. and McHugh, S. 2004. Selectable Marker Genes in Transgenic Plants: Applications, Alternatives and Biosafety. J. Biotechnol., 107: 193-232.
18. Manderscheid, R. and Wild, A. 1986. Studies on the Mechanism of Inhibition by Phosphinothricin of Glutamine Synthetase Isolated from Triticum aestivum L. J. Plant Physiol., 123: 135-142.
19. Nandula, V. K., Reddy, K. N., Duke, S. O. and Poston, D. H. 2005. Glyphosate-resistant Weeds: Current Status and Future Outlook. Outlooks Pest Manag., 16: 183-187.
20. Neve, P. 2007. Challenges for Herbicide Resistance Evolution and Management: 50 Years after Harper. Weed Res., 47(5): 365-369.
21. Peng, H. F., Zhang, Z. F., Wu, B., Chen, X. H., Zhang, G. Q., Zhang, Z. M., Wan, B. H. and Lu, Y. P. 2008. Molecular Mapping of Two Reverse Photoperiod-sensitive Genic Male Sterility Genes (rpms1 and rpms2) in Rice (Oryza sativa L.). Theor. Appl. Genet., 118: 77-83.
22. Peng, R. H., Yao, Q. H., Xiong, A. S., Cheng, Z. M. and Li, Y. 2006. Codon-modifications and An Endoplasmic Reticulum-targeting Sequence Additively Enhance Expression of An Aspergillus Phytase Gene in Transgenic Canola. Plant Cell Rep., 25(2): 124-132.
23. Pline-Srnic, W. 2006. Physiological Mechanisms of Glyphosate Resistance. Weed Techn., 20(2): 290-300.
24. Reddy, K. N., Zablotowicz, R. M., Bellaloui, N. and Ding, W. 2011. Glufosinate Effects on Nitrogen Nutrition, Growth, Yield, and Seed Composition in Glufosinate-resistant and Glufosinate-sensitive Soybean. Int. J. Agron., 2011:1-9.
25. Sambrook, J. and Russell, D. W. 2001. Molecular Cloning: A Laboratory Manual. 3rd Edition, New York, PP: 492-502.
26. Smart, C. C. and Amrhein, N. 1987. Ultrastructural Localisation by Protein A-gold Immunocytochemistry of 5-enolpyruvylshikimic-3-phosphatesynthase in a Plant Cell Culture which Overproduces the Enzyme. Planta, 170: 1-6.
27. Thompson, C. J., Movva, N. R., Tichard, R., Crameri, R., Davies, J. E., and Lauwereys, M. 1987. Characterization of the Herbicide-resistance Gene Bar from Streptomyces hygroscopicus. EMBO J., 6(9): 2519-2523.
28. Thompson, G. A., Hiatt, W. R., Facciotti, D., Stalker, D. M. and Comai, L. 1987. Expression in Plants of a Bacterial Gene Coding for Glyphosate Resistance. Weed Sci., 35(Suppl. 1): 19-23.
29. Toki, S., Hara, N., Ono, K., Onodera, H., Tagiri, A., Oka, S. and Tanaka, H. 2006. Early Infection of Scutellum Tissue with Agrobacterium Allows High-speed Transformation of Rice. The Plant J., 47: 969-976.
30. USDA-APHIS. 1999. Environmental Assessment for Petition 98-329-01p. Determination of Non-regulated Status for Rice Genetically Engineered for Glufosinate Herbicide Tolerance. http://www.aphis.usda.gov/brs/aphisdocs2/98_32901p_com.pdf
31. Wang, X. J., Lang, Z. H., Lu, W., Lin, M., Shan, A. S. and Huang, D. F. 2008. Transferring Glyphosate-tolerant mG2-epsps Gene into Maize Inbred Line via Pollen-tube Pathway. J. Agric. Sci. Techn., 10(4): 56-62.
32. Wang, H. Y., Li, Y. F., Xie, L. X. and Xu P. L. 2003. Expression of a Bacterial aroA Mutant, aroA-M1, Encoding 5-enolpyruvylshikimate-3-phosphate Synthase for the Production of Glyphosate-resistant Tobacco Plants. J. Plant Res., 116:455-460.
33. Xiao, G. Y. 1997. The View of Crop Herbicide Resistance for Heterosis Utilization. Hybrid rice, 12(5): 1-3.
34. Xiao, G. Y., Yuan, L. P. and Sun, S. S. M. 2007. Strategy and Utilization of a Herbicide Resistance Gene in Two-line Hybrid Rice. Mol. Breeding., 20: 287-292.
35. Yasuor, H., Abu-Abied, M., Belausov, E., Madmony, A., Sadot, E., Riov, J. and Rubin, B. 2006. Glyphosate-induced Anther Indehiscence in Cotton is Partially Temperature Dependent and Involves Cytoskeleton and Secondary Wall Modifications and Auxin Accumulation. Plant Physiol., 141(4): 1306-1315.
36. Zhou, H., Liu, Q., Li, J., Jiang, D., Zhou, L., Wu, P., Lu, S., Li, F., Zhu, L. and Liu, Z. 2012. Photoperiod- and Thermo-sensitive Genic Male Sterility in Rice are Caused by a Point Mutation in a Novel Noncoding RNA that Produces a Small RNA. Cell Res., 22(4): 649-60.
Journal of Agricultural Science and Technology
Volume 16, Issue 1
January and February 2014
Pages 217-228