Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic <i>Arabidopsis</i>
Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been a...
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MDPI AG
2021-05-01
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| author | Chen Yang Jia Ge Xiaokang Fu Keming Luo Changzheng Xu |
| author_facet | Chen Yang Jia Ge Xiaokang Fu Keming Luo Changzheng Xu |
| author_sort | Chen Yang |
| collection | DOAJ |
| description | Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic <i>Arabidopsis</i> containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two <i>loxP</i> sites, including promoters, resistance genes, reporter genes, and split-<i>Cre</i> genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which <i>NCre</i> was driven by <i>proDD45</i>, and <i>CCre</i> was driven by <i>proACA9</i> and <i>proDLL</i>. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the <i>NCre</i> and <i>CCre</i> genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns. |
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| spelling | doaj.art-10828934614e46749e4c795b7bf5cdbb2023-11-21T19:12:04ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-05-012210508010.3390/ijms22105080Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic <i>Arabidopsis</i>Chen Yang0Jia Ge1Xiaokang Fu2Keming Luo3Changzheng Xu4Chongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, Key Laboratory of Eco-Environments of Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing 400715, ChinaChongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, Key Laboratory of Eco-Environments of Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing 400715, ChinaChongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, Key Laboratory of Eco-Environments of Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing 400715, ChinaChongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, Key Laboratory of Eco-Environments of Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing 400715, ChinaChongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, Key Laboratory of Eco-Environments of Three Gorges Reservoir Region, Ministry of Education, School of Life Sciences, Southwest University, Chongqing 400715, ChinaGenetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic <i>Arabidopsis</i> containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two <i>loxP</i> sites, including promoters, resistance genes, reporter genes, and split-<i>Cre</i> genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which <i>NCre</i> was driven by <i>proDD45</i>, and <i>CCre</i> was driven by <i>proACA9</i> and <i>proDLL</i>. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the <i>NCre</i> and <i>CCre</i> genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.https://www.mdpi.com/1422-0067/22/10/5080<i>Arabidopsis</i>biosafetyCre/LoxPhybridreproductive cell specificity |
| spellingShingle | Chen Yang Jia Ge Xiaokang Fu Keming Luo Changzheng Xu Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic <i>Arabidopsis</i> International Journal of Molecular Sciences <i>Arabidopsis</i> biosafety Cre/LoxP hybrid reproductive cell specificity |
| title | Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic <i>Arabidopsis</i> |
| title_full | Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic <i>Arabidopsis</i> |
| title_fullStr | Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic <i>Arabidopsis</i> |
| title_full_unstemmed | Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic <i>Arabidopsis</i> |
| title_short | Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic <i>Arabidopsis</i> |
| title_sort | dual reproductive cell specific promoter mediated split cre loxp system suitable for exogenous gene deletion in hybrid progeny of transgenic i arabidopsis i |
| topic | <i>Arabidopsis</i> biosafety Cre/LoxP hybrid reproductive cell specificity |
| url | https://www.mdpi.com/1422-0067/22/10/5080 |
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