Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna |
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| Institution: | 1. Chongqing Key Laboratory of Plant Resource Conservation and Germplasm Innovation, SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing, 400715, China;2. Chongqing Academy of Science and Technology, Chongqing, 401123, China;3. College of Life Sciences, Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang, 550025, China;4. TAAHC-SWU Medicinal Plant Joint R&D Centre, Xizang Agricultural and Husbandry College, Nyingchi of Tibet, 860000, China;5. College of Pharmaceutical Sciences, Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Ministry of Education), Southwest University, Chongqing, 400715, China;6. Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences, Chongqing, 400712, China;1. National Bioenergy Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA;2. Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM, 87545, USA;3. Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA;1. Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Republic of Korea;2. College of Pharmacy, Seoul National University, Seoul, 08826, Republic of Korea;3. Department of Biomedical Sciences, Seoul National University, College of Medicine, Seoul, Republic of Korea;4. Department of Genetics, Yale Stem Cell Center, Child Study Center, Interdepartmental Neuroscience Program, Yale School of Medicine, New Haven, CT, 06520, USA;5. Gachon Institute of Pharmaceutical Sciences, College of Pharmacy, Gachon University, Incheon, 21936, Republic of Korea;6. Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang, 37673, Republic of Korea;7. Center for Self-assembly and Complexity, Institute for Basic Science (IBS), Pohang, 37673, Republic of Korea;1. Department of Microbiology and Molecular Genetics, University of California, Davis. One Shields Avenue, Davis, CA, 95616, USA;2. Department of Biomedical Engineering, University of California, Davis. One Shields Avenue, Davis, CA, 95616, USA;1. School of Biosciences, University of Sheffield, Sheffield, S10 2TN, UK;2. Sustainable Process Technologies, Faculty of Engineering, University of Nottingham, Nottingham, NG7 2RD, UK;3. Department of Biology, University of York, York, YO10 5DD, UK;4. School of Life Sciences, University of Nottingham, QMC, Nottingham, NG7 2UH, UK;5. Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, UK;6. biOMICS Mass Spectrometry Facility, Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, UK;7. Chemical Engineering, School for Engineering of Matter, Transport, and Energy, Arizona State University, Tempe, AZ, 85287-6106, USA;1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea;2. KI for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea;1. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China;2. College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China |
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| Abstract: | Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs’ production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost. |
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| Keywords: | Calmodulin Genetic engineering Glyphosate resistance Transgenic homozygous lines Tropane alkaloids |
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