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Open AccessArticle

Heat-Shock-Induced Removal of Transgenes Using the Gene-Deletor System in Hybrid Aspen (Populus tremula × P. tremuloides)

by Beibei Wang 1,2,†, Yan Zhang 1,†, Jian Zhao 1, Mingliang Dong 1 and Jinfeng Zhang 1,*
1
Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, National Engineering Laboratory for Tree Breeding, Key Laboratory of Genetics and Breeding in Forest Tree and Ornamental Plants of Ministry of Education, Key Laboratory of Forest Trees and Ornamental Plants Biological Engineering of State Forestry Administration, College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China
2
Beijing Academy of Forestry and Pomology Sciences, Beijing 100093, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Genes 2018, 9(10), 484; https://doi.org/10.3390/genes9100484
Received: 26 July 2018 / Revised: 23 September 2018 / Accepted: 1 October 2018 / Published: 8 October 2018
(This article belongs to the Section Plant Genetics and Genomics)
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. View Full-Text
Keywords: gene-deletor system; Populus; heat shock; methylation; expression gene-deletor system; Populus; heat shock; methylation; expression
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Wang, B.; Zhang, Y.; Zhao, J.; Dong, M.; Zhang, J. Heat-Shock-Induced Removal of Transgenes Using the Gene-Deletor System in Hybrid Aspen (Populus tremula × P. tremuloides). Genes 2018, 9, 484.

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