Abstract
To evaluate the efficacy of the gene-deletor system in aspen, we evaluated the system for foreign gene removal in a hybrid aspen clone, INRA 353-53 (Populus tremula × P. tremuloides). The recombinase flipping DNA (FLP) gene was under the control of the heat-inducible promoter of Gmhsp17.6-L, and the β-glucuronidase (gusA) gene which was under the control of the 35S promoter and were constructed using the gene-deletor system in the pCaLFGmFNLFG vector. Six transgenic plants and their sublines were heated at 42 °C for 8 h and gene deletion was verified by polymerase chain reaction (PCR). Three lines exhibited partial transgene deletion while the remaining three lines did not delete. Transgenic lines were evaluated by Southern-blot analyses, verifying that the six transgenic plant lines all had a single copy of transfer DNA (t-DNA). Two partial-deletion lines and two non-deletion lines were analysed for methylation and expression of promoter and recombinase. Hardly any methylation was detected in the Gmhsp17.6-L promoter or recombinase FLP gene sequences, however, the expression of the promoter and recombinase was increased significantly in the partial-deletion compared with the non-deletion line after heat-shock treatment. These results suggest that the excision efficiency had no direct relationship with methylation status of the Gmhsp17.6-L promoter and FLP recombinase, yet was affected by the expression of the Gmhsp17.6-L and FLP after heat-shock treatment.
Highlights
Since the first report of transgenic aspen [1], researchers have been using transgenic technology to improve plant biomass and increase resistance to insects, herbicides, and disease in aspen [2,3,4].Transgenic plants can be used to address scientific questions by modification of flowering, hormone levels, and phytoremediation
The pCaLFGmFNLFG vector was constructed as follows: the NOS and RB sequences were amplified from the pGM626-ZMF-ABt vector using the III-NOS-FF/III-NOS-RR and III-RB-FF/III-RB-R primers (Table S1) and ligated to the nopaline synthase and right border (NOS-RB) sequence generated by overlap polymerase chain reaction (PCR) via the KpnI and SphI restriction sites
Putative transgenic aspen obtained using Agrobacterium-mediated transformation methods were verified for transgenicity by PCR using the GUSF/GUSR primers for gusA and GmHSP-F/GmHSP-R
Summary
Since the first report of transgenic aspen [1], researchers have been using transgenic technology to improve plant biomass and increase resistance to insects, herbicides, and disease in aspen [2,3,4]. Transgenic plants may affect gene flow, increasing public concerns about environmental safety and thereby limiting the use of transgenic technology in trees. Various strategies have been developed to remove marker or foreign genes in genetically modified plants. These methods include co-transformation of two transfer DNA (t-DNA) regions [5], homologous recombination [6], transposon-mediated reposition [7], site-specific recombination [8,9,10], and the gene-deletor system [11,12,13].
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