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.

Inteins—mechanism of protein splicing, emerging regulatory roles, and applications in protein engineering

“…In this case, each segment of an herbicide resistance protein is fused to a split intein, and the two segments are inserted into different plant chromosomes. Under these conditions, co-inheritance of both segments is significantly reduced relative to the single-gene approach (Wang et al, 2014).…”

Gene Flow From Major Genetically Modified Crops and Strategies for Containment and Mitigation of Transgene Escape: A Review

“…Dong et al (2015) proposed a containment strategy taking intein as major working agents, they proposed to split the gene of interest into two different sequences and place far away in genome and combine them with inteins, as far away sequences have more chances to segregate during meiosis and named it as gene splitting. Wang et al (2014) to produce GM tobacco. This technique showed no signs of transgenes into F1 population but backcross population showed 25% plants with trans-gene.…”

The superwoman1‐cleistogamy2 mutant is a novel resource for gene containment in rice

“…Various strategies have been designed, such as transgene removal from pollen by molecular excision, transgene splitting using intein flanking sequences, male sterility or generation of cleistogamous (i.e. self‐pollinating, closed‐flower) plants to prevent pollen dispersal (Moon et al ., ; Ohmori et al ., ; Shinoyama et al ., ; Toppino et al ., ; Wang et al ., ). In rice, the cleistogamy trait can be introduced by modification of a single gene.…”