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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.

SUBMITTER: Wang XJ

PROVIDER: S-EPMC4051838 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Gene-splitting technology: a novel approach for the containment of transgene flow in Nicotiana tabacum.

Wang Xu-Jing XJ Jin Xi X Dun Bao-Qing BQ Kong Ning N Jia Shi-Rong SR Tang Qiao-Ling QL Wang Zhi-Xing ZX

PloS one 20140610 6

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) ...[more]

PMID: 24915192

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Project description:BACKGROUND:Heat shock proteins 90 (HSP90s) are a highly conserved protein family of cellular chaperones widely found in plants; they play a fundamental role in response to biotic and abiotic stresses. The genome-wide analysis of HSP90 gene family has been completed for some species; however, it has been rarely reported for the tobacco HSP90 genes. RESULTS:In this study, we systematically conducted genome-wide identification and expression analysis of the tobacco HSP90 gene family, including gene structures, evolutionary relationships, chromosomal locations, conserved domains, and expression patterns. Twenty-one NtHSP90s were identified and classified into eleven categories (NtHSP90-1 to NtHSP90-11) based on phylogenetic analysis. The conserved structures and motifs of NtHSP90 proteins in the same subfamily were highly consistent. Most NtHSP90 proteins contained the ATPase domain, which was closely related to conserved motif 2. Motif 5 was a low complexity sequence and had the function of signal peptide. At least 6 pairs of NtHSP90 genes underwent gene duplication, which arose from segment duplication and tandem duplication events. Phylogenetic analysis showed that most species expanded according to their own species-specific approach during the evolution of HSP90s. Dynamic expression analysis indicated that some NtHSP90 genes may play fundamental roles in regulation of abiotic stress response. The expression of NtHSP90-4, NtHSP90-5, and NtHSP90-9 were up-regulated, while NtHSP90-6, and NtHSP90-7 were not induced by ABA, drought, salt, cold and heat stresses. Among the five treatments, NtHSP90s were most strongly induced by heat stress, and weakly activated by ABA treatment. There was a similar response pattern of NtHSP90s under osmotic stress, or extreme temperature stress. CONCLUSIONS:This is the first genome-wide analysis of Hsp90 in N. tabacum. These results indicate that each NtHSP90 member fulfilled distinct functions in response to various abiotic stresses.

Dataset Information

Gene-splitting technology: a novel approach for the containment of transgene flow in Nicotiana tabacum.

Publications

Gene-splitting technology: a novel approach for the containment of transgene flow in Nicotiana tabacum.

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