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Identification of Herbicide-tolerant Gene aroAA1501 Split Site |
SONG Ya-Ya, YANG Jiang-Tao, WANG Zhi-Xing*, WANG Xu-Jing* |
Key Laboratory of Safety Evaluation (Molecular) of Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Sciences, Institute of Biotechnology, Chinese Academy of Agricultural Sciences, Beijing 100081, China |
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Abstract Gene splitting technology can effectively avoid the escape of target traits in genetically modified crops to the environment. In order to cultivate a new glyphosate-resistant aroAA1501 transgenic rice (Oryza sativa) using gene resolution technology, 13 possible resolution sites were screened out based on the advanced structure of aroAA1501 protein in this study. aroAA1501 was split into N-terminal (An) and C-terminal (Ac) by PCR, and is combined with N-terminal (In) and C-terminal (Ic) of SspDnaE intein to form fusion genes An-In and Ic-Ac. Based on pETDuet-1, a total of 40 prokaryotic expression vectors containing An-In, Ic-Ac and An-In / Ic-Ac were constructed. The constructed prokaryotic expression vector was introduced into the auxotrophic Escherichia coli strain ER2799. It was proved by functional complementation tests that aroAA1501 was split at the positions 141/142, 224/225, and 230/231. The protein was reassembled into a functional intact protein mediated by the protein intein. The glyphosate tolerance test proved that 230/231 was the most suitable resolving site. The reassembled protein after resolving was 3 times more tolerant to glyphosate than the complete aroAA1501 protein. This study provides basic data for the later cultivation of glyphosate-resistant transgenic rice with aroAA1501 gene.
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Received: 19 September 2019
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Corresponding Authors:
* wangcotton@126.com; wangxujing@caas.cn
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[1] 董玉凤, 王旭静, 宋亚亚, 等. 2019. 利用基因拆分技术培育耐草甘膦转基因水稻的研究[J]. 作物学报, 45(3): 344-353. (Dong Y F, Wang X J, Song Y Y,et al.2019. Study on the cultivation of glyphosate-tolerant transgenic rice by gene resolution technology[J]. Acta Agronomica Sinica, 45(3): 344-353.) [2] 李亮, 梁爱敏, 李鑫鑫, 等. 2008.斯式假单胞菌A1501EPSP合酶Asn130位点抗草甘膦机理研究[C]. 2008年中国微生物学会学术年会. (Li L, Liang A M, Li X X, et at.2008. Study on the mechanism of anti-glyphosate in Asn130 site of Pseudomonas syringae A1501 EPSP synthase[C]. 2008 Annual Meeting of Chinese Society of Microbiology. [3] Carvajal-Vallejos P, Pallissé R, Mootz H D, et al.2012. Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources[J]. Journal of Biological Chemistry, 287(34): 28686-28696. [4] Chen L, Pradhan S, Evans T C Jr.2001. Herbicide resistance from a divided EPSPS protein: The split Synechocystis DnaE intein as an in vivo affinity domain[J]. Gene, 263(1-2): 39-48. [5] Chin H G, Kim G D, Marin I, et al.2003. Protein trans-splicing in transgenic plant chloroplast: Reconstruction of herbicide resistance from split genes[J]. Proceedings of the National Academy of Sciences of the USA, 100(8): 4510-4515. [6] Choi J J, Nam K H, Min B, et al.2006. Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans[J]. Journal of Molecular Biology, 356(5): 1093-1106. [7] Dun B Q, Wang X J, Lu W, et al.2007. Reconstitution of glyphosate resistance from a split 5-enolpyruvyl shikimate-3-phosphate synthase gene in Escherichia coli and transgenic tobacco[J]. Applied and Environmental Microbiology, 73(24): 7997-8000. [8] Elleuche S, Pöggeler S.2010. Inteins, valuable genetic elements in molecular biology and biotechnology[J]. Applied Microbiology and Biotechnology, 87(2): 479-489. [9] Evans Jr T J, Xu M Q, Pradhan S.2005. Protein splicing elements and plants: From transgene containment to protein purification[J]. Annual Review of Plant Biology, 56: 375-392. [10] Ge J, Wan L, Yang C, et al.2016. Inteinmediated Cre protein assembly for transgene excision in hybrid progeny of transgenic Arabidopsis[J]. Plant Cell, 35(10): 2045-2053. [11] Han X, Han F, Ren X, et al.2013. Ssp DnaE split-intein mediated split-Cre reconstitution in tobacco[J]. Plant Cell Tissue & Organ Culture, 113(3): 529-542. [12] Jim M. Dunwell and Andy C. Wetten (eds.). 2012. Transgenic Plants: Methods and Protocols, Methods in Molecular Biology[J]. vol. 847. [13] Kane P M, Yamashiro C T, Wolczyk D F, et al.1990. Protein splicing converts the yeast TFP1 gene product to the 69‐kD subunit of the vacuolar H(+)‐adenosine triphosphatase[J]. Science, 250(4981): 651-657. [14] Kempe K, Rubtsova M, Gils M.2009. Intein-mediated protein assembly in transgenic wheat: Production of active barnase and acetolactate synthase from split genes[J]. Plant Biotechnology Journal. J, 7: 283-97. [15] Kempe K, Rubtsova M, Gils M.2014. Split-gene system for hybrid wheat seed production[J]. Proceedings of the National Academy of Sciences of the USA, 111(25): 9097-102. [16] Mills K V, Johnson M A, Perler F B.2014. Protein splicing: How inteins escape from precursor proteins[J]. Journal of Biological Chemistry, 289(21): 14498-14505. [17] Shah N H, Muir T W.2014. Inteins: Nature's gift to protein chemists Chem[J]. Science, 5(1): 446-461. [18] Sun L, Ghosh I, Paulus H, et al.2001. Protein trans-splicing to produce herbicide-resistant acetolactate synthase[J]. Applied and Environmental Microbiology, 67(3): 1025-1029. [19] Volkmann G, Mootz H D.2013. Recent progress in intein research: From mechanism to directed evolution and applications Cell[J]. Cellular and Molecular Life Sciences, 70(7): 1185-1206. [20] Wang X J, Jin X, Dun B Q, et al.2014. Gene-Splitting Technology: A Novel Approach for the Containment of Transgene Flow in Nicotiana tabacum[J]. Plos One [21] Wu H, Hu Z, Liu X Q.1998. Protein trans-splicing by a split intein encoded in a split DnaE gene ofSynechocystis sp. PCC6803[J]. Proceedings of the National Academy of Sciences of the USA, 95(16): 9226-9231. [22] Wei X Y, Sakr S, Li J H,et al.2006. Expression of split dnaE genes andtrans-splicing of DnaE intein in the developmental cyanobacterium Anabaena sp. PCC 7120[J]. Research in Microbiology, 157(3): 227-234. [23] Zettler J, Schutz V, Mootz H D.2009. The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction[J]. Febs Letters, 583(5): 909-914. [24] Yang J J, George C, Tina V, et al.2003. Intein-mediated assembly of a functional β-glucuronidase in transgenic plants[J]. Proceedings of the National Academy of Sciences of the USA, 100(6): 3513-3518. |
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