title: Gene-splitting technology: a novel approach for the containment of transgene flow in Nicotiana tabacum.
abstract: 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.
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Xu-Jing
authors.initials: XJ
authors.lastname: Wang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Jin
authors.affiliation: National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Bao-Qing
authors.initials: BQ
authors.lastname: Dun
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Kong
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Shi-Rong
authors.initials: SR
authors.lastname: Jia
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Qiao-Ling
authors.initials: QL
authors.lastname: Tang
authors.affiliation: Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
authors.forename: Zhi-Xing
authors.initials: ZX
authors.lastname: Wang
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000235:genetics; D015153:Blotting, Western; D051456:Gene Flow; D005821:Genetic Techniques; D005998:Glycine / Q000031:analogs & derivatives / Q000633:toxicity; D053209:Herbicide Resistance / Q000235:genetics; D006720:Homozygote; D047668:Inteins / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development; D020040:Trans-Splicing; D014170:Transformation, Genetic; D019076:Transgenes / Q000235:genetics*; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D005998:Glycine; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000097797:Glyphosate