Tan Quang Tu1, Phat Tien Do2,3, Doai Van Nguyen2, Nhan Thi Thanh Pham4, Tam Thi Nguyen1, Mau Hoang Chu5. 1. TNU- University of Education, Thai Nguyen, 250000, Vietnam. 2. VAST- Institute of Biotechnology, Ha Noi, 100000, Vietnam. 3. VAST - Graduate University of Sciences and Technology, Hanoi, 100000, Vietnam. 4. TNU- University of Education, Thai Nguyen, 250000, Vietnam. ptnhanbio@tnue.edu.vn. 5. TNU- University of Education, Thai Nguyen, 250000, Vietnam. chuhoangmau@tnu.edu.vn.
Abstract
BACKGROUND: In the alkaloid biosynthetic pathways of Stephania and Rannunculaceae, columbamine O-methyltransferase (CoOMT) is an important enzyme that catalyses the formation of the tetrahydropalmatin (rotundin) biosynthesis pathway. In this study, the transgenic construct pBI121-35S-CoOMT-cmyc-Kdel was designed successfully. METHODS AND RESULTS: The real-time RT-PCR results proved that the CoOMT transgene was successfully introduced into Nicotiana tabacum L. plants and produced mRNA. Its transcription levels in three transgenic tobacco lines, T0-7, T0-9, and T0-20, in the T0 generation were higher than those in wild-type tobacco plants. By analysing Western blots and ELISAs, three T0 generation transgenic tobacco lines also expressed recombinant CoOMT (rCoOMT) protein with a molecular weight of approximately 40 kDa, and its contents ranged from 0.048 μg mg-1 to 0.177 μg mg-1. These data illustrated that the CoOMT transgene was expressed; thus, the rCoOMT protein synthesis efficiency increased significantly in comparison with that of the wild-type tobacco plants. The total alkaloid contents ranged from 2.12 g 100 g-1 (of dry weight) to 3.88 g 100 g-1 (of dry weight). The T0-20 plant had the highest total alkaloid content (3.88 g 100 g-1 of dry weight), followed by the T0-7 line (2.75 g 100 g-1 of dry weight). The total alkaloid contents of the CoOMT transgenic tobacco lines increased by approximately 1.09-1.83-fold compared to the wild-type tobacco plants. CONCLUSIONS: This is the first study on the transformation and expression of the CoOMT gene in N. tabacum plants. Initial results of the analysis of transgenic plants proved that the transgenic structure pBI121- CoOMT-Cmyc-Kdel can be used for transformation into Stephania plants.
BACKGROUND: In the alkaloid biosynthetic pathways of Stephania and Rannunculaceae, columbamine O-methyltransferase (CoOMT) is an important enzyme that catalyses the formation of the tetrahydropalmatin (rotundin) biosynthesis pathway. In this study, the transgenic construct pBI121-35S-CoOMT-cmyc-Kdel was designed successfully. METHODS AND RESULTS: The real-time RT-PCR results proved that the CoOMT transgene was successfully introduced into Nicotiana tabacum L. plants and produced mRNA. Its transcription levels in three transgenic tobacco lines, T0-7, T0-9, and T0-20, in the T0 generation were higher than those in wild-type tobacco plants. By analysing Western blots and ELISAs, three T0 generation transgenic tobacco lines also expressed recombinant CoOMT (rCoOMT) protein with a molecular weight of approximately 40 kDa, and its contents ranged from 0.048 μg mg-1 to 0.177 μg mg-1. These data illustrated that the CoOMT transgene was expressed; thus, the rCoOMT protein synthesis efficiency increased significantly in comparison with that of the wild-type tobacco plants. The total alkaloid contents ranged from 2.12 g 100 g-1 (of dry weight) to 3.88 g 100 g-1 (of dry weight). The T0-20 plant had the highest total alkaloid content (3.88 g 100 g-1 of dry weight), followed by the T0-7 line (2.75 g 100 g-1 of dry weight). The total alkaloid contents of the CoOMT transgenic tobacco lines increased by approximately 1.09-1.83-fold compared to the wild-type tobacco plants. CONCLUSIONS: This is the first study on the transformation and expression of the CoOMT gene in N. tabacum plants. Initial results of the analysis of transgenic plants proved that the transgenic structure pBI121- CoOMT-Cmyc-Kdel can be used for transformation into Stephania plants.