The potential impact of transgene escape on the environment and food safety is a major concern to the scientists and public. This work aimed to assess the effect of intein-mediated gene splitting on containment of transgene flow. Two fusion genes, EPSPSn-In and Ic-EPSPSc, were constructed and integrated into N. tabacum, using Agrobacterium tumefaciens-mediated transformation. EPSPSn-In encodes the first 295 aa of the herbicide resistance gene 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) fused with the first 123 aa of the Ssp DnaE intein (In), whereas Ic-EPSPSc encodes the 36 C-terminal aa of the Ssp DnaE intein (Ic) fused to the rest of EPSPS C terminus peptide sequences. Both EPSPSn-In and Ic-EPSPSc constructs were introduced into the same N. tabacum genome by genetic crossing. Hybrids displayed resistance to the herbicide N-(phosphonomethyl)-glycine (glyphosate). Western blot analysis of protein extracts from hybrid plants identified full-length EPSPS. Furthermore, all hybrid seeds germinated and grew normally on glyphosate selective medium. The 6-8 leaf hybrid plants showed tolerance of 2000 ppm glyphosate in field spraying. These results indicated that functional EPSPS protein was reassembled in vivo by intein-mediated trans-splicing in 100% of plants. In order to evaluate the effect of the gene splitting technique for containment of transgene flow, backcrossing experiments were carried out between hybrids, in which the foreign genes EPSPSn-In and Ic-EPSPSc were inserted into different chromosomes, and non-transgenic plants NC89. Among the 2812 backcrossing progeny, about 25% (664 plantlets) displayed glyphosate resistance. These data indicated that transgene flow could be reduced by 75%. Overall, our findings provide a new and highly effective approach for biological containment of transgene flow.
References
[1]
James C (2013) Global stutus of commercialized biotech/GM crops: 2012. China Biotech 33: 1–8.
[2]
Ruf S, Karcher D, Bock R (2007) Determining the transgene containment level provided by chloroplast transformation. Proc Natl Acad Sci U S A 104: 6998–7002. doi: 10.1073/pnas.0700008104
[3]
Kempken F (2010) Engineered Male Sterility. Biotechnology in Agriculture and Forestry 64: 253–265. doi: 10.1007/978-3-642-02391-0_14
[4]
Gidoni D, Srivastava V, Carmi N (2008) Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants. In Vitro Cellular & Developmental Biology-Plant 44: 457–467. doi: 10.1007/s11627-008-9140-3
[5]
Benitez ER, Khan NA, Matsumura H, Abe J, Takahashi R (2010) Varietal differences and morphology of cleistogamy in soybean. Crop Science 50: 185–190. doi: 10.2135/cropsci2009.02.0108
[6]
Munsch M, Camp KH, Stamp P, Weider C (2008) Modern maize hybrids can improve grain yield as plus-hybrids by the combined effects of cytoplasmic male sterility and allo-pollination. Maydica 53: 262–268.
[7]
Svab Z, Maliga P (2007) Exceptional transmission of plastids and mitochondria from the transplastomic pollen parent and its impact on transgene containment. Proc Natl Acad Sci U S A 104: 7003–7008. doi: 10.1073/pnas.0700063104
[8]
Yoshida H, Itoh J, Ohmori S, Miyoshi K, Horigome A, et al. (2007) superwoman1-cleistogamy, a hopeful allele for gene containment in GM rice. Plant Biotechnol J 5: 835–846. doi: 10.1111/j.1467-7652.2007.00291.x
[9]
Leflon M, Hüsken A, Njontie C, Kightley S, Pendergrast D, et al. (2009) Stability of the cleistogamous trait during the flowering period of oilseed rape. Plant breeding 129: 13–18. doi: 10.1111/j.1439-0523.2009.01645.x
[10]
Al-Ahmad H, Galili S, Gressel J (2005) Poor competitive fitness of transgenically mitigated tobacco in competition with the wild type in a replacement series. Planta 222: 372–385. doi: 10.1007/s00425-005-1540-6
[11]
Moon HS, Li Y, Stewart CN Jr (2010) Keeping the genie in the bottle: transgene biocontainment by excision in pollen. Trends Biotechnol 28: 3–8. doi: 10.1016/j.tibtech.2009.09.008
[12]
Husken A, Prescher S, Schiemann J (2010) Evaluating biological containment strategies for pollen-mediated gene flow. Environ Biosafety Res 9: 67–73. doi: 10.1051/ebr/2010009
[13]
Elleuche S, Poggeler S (2010) Inteins, valuable genetic elements in molecular biology and biotechnology. Appl Microbiol Biotechnol 87: 479–489. doi: 10.1007/s00253-010-2628-x
[14]
Hirata R, Ohsumk Y, Nakano A, Kawasaki H, Suzuki K, et al. (1990) Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem 265: 6726–6733.
[15]
Kane PM, Yamashiro CT, Wolczyk DF, Neff N, Goebl M, et al. (1990) Protein splicing converts the yeast TFP1 gene product to the 69-kD subunit of the vacuolar H(+)-adenosine triphosphatase. Science 250: 651–657. doi: 10.1126/science.2146742
[16]
Perler FB (2002) InBase: the Intein Database. Nucleic Acids Res 30: 383–384. doi: 10.1093/nar/30.1.383
[17]
Wu H, Hu Z, Liu XQ (1998) Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc Natl Acad Sci U S A 95: 9226–9231. doi: 10.1073/pnas.95.16.9226
[18]
Chin HG, Kim GD, Marin I, Mersha F, Evans TC Jr, et al. (2003) Protein trans-splicing in transgenic plant chloroplast: reconstruction of herbicide resistance from split genes. Proc Natl Acad Sci U S A 100: 4510–4515. doi: 10.1073/pnas.0736538100
[19]
Yang J, Fox GC Jr, Henry-Smith TV (2003) Intein-mediated assembly of a functional beta-glucuronidase in transgenic plants. Proc Natl Acad Sci U S A 100: 3513–3518. doi: 10.1073/pnas.0635899100
[20]
Gils M, Rubtsova M, Kempe K (2012) Split-transgene expression in wheat. Methods Mol Biol 847: 123–135. doi: 10.1007/978-1-61779-558-9_11
[21]
Zhu Y, Yu ZL, Lin M (2003) Bioresistance and biodegradation of glyphosate and construction of transgenic plants. Molecular Plant Breeding 1: 435–441.
[22]
Dun BQ, Wang XJ, Lu W, Zhao ZL, Hou SN, et al. (2007) Reconstitution of glyphosate resistance from a split 5-enolpyruvyl shikimate-3-phosphate synthase gene in Escherichia coli and transgenic tobacco. Appl Environ Microbiol 73: 7997–8000. doi: 10.1128/aem.00956-07
[23]
Dafny-Yelin M, Chung SM, Frankman EL, Tzfira T (2007) pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants. Plant Physiol 145: 1272–1281. doi: 10.1104/pp.107.106062
[24]
Khan MS, Khalid AM, Malik KA (2005) Intein-mediated protein trans-splicing and transgene containment in plastids. Trends Biotechnol 23: 217–220. doi: 10.1016/j.tibtech.2005.03.006
[25]
Bock R, Khan MS (2004) Taming plastids for a green future. Trends Biotechnol 22: 311–318. doi: 10.1016/s0167-7799(04)00079-4
[26]
Iwai H, Zuger S, Jin J, Tam PH (2006) Highly efficient protein trans-splicing by a naturally split DnaE intein from Nostoc punctiforme. FEBS Lett 580: 1853–1858. doi: 10.1016/j.febslet.2006.02.045
[27]
Rieben S, Kalinina O, Schmid B, Zeller SL (2011) Gene flow in genetically modified wheat. PLoS One 6: e29730. doi: 10.1371/journal.pone.0029730
[28]
Heuberger S, Ellers-Kirk C, Tabashnik BE, Carriere Y (2010) Pollen- and seed-mediated transgene flow in commercial cotton seed production fields. PLoS One 5: e14128. doi: 10.1371/journal.pone.0014128
[29]
Dyer GA, Serratos-Hernandez JA, Perales HR, Gepts P, Pineyro-Nelson A, et al. (2009) Dispersal of transgenes through maize seed systems in Mexico. PLoS One 4: e5734. doi: 10.1371/journal.pone.0005734
