Confirmation of ‘Pollen- and Seed-Specific Gene Deletor’ System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions
Abstract
:1. Introduction
2. Results
2.1. Gene Deletor Vector Construction
2.2. Production and Confirmation of N. tabacum Transgenic Lines
2.3. The ‘Gene Deletor’ System Excised Transgenes from the Pollen and Seeds of Greenhouse-Grown N. Tabacum
2.4. The ‘Gene Deletor’ System Stably and Completely Excised Transgenes from the Pollen and Seeds Produced by Transgenic N. tabacum Grown in the Field
2.5. Molecular Analysis of Transgene Excision from Pollen and Seeds
3. Discussion
4. Materials and Methods
4.1. Nicotiana tabacum L. Transformation Using the ‘Gene Deletor’ Vector
4.2. GUS Histochemical Staining
4.3. PCR Detection of Transgenes
4.4. Detection of Transgene Excision Efficiency in Greenhouse-Grown Transgenic Plants
4.5. Confirmation of Transgene Excision in the T1 Generation of Greenhouse-Grown Transgenic Plants
4.6. Estimation of Transgene Copy Numbers in Transgenic Plants
4.7. Transgene Excision in Field-Grown Transgenic Plants
4.8. Molecular Verification of Transgene Excision in the Transgenic Plants
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Transformation | Constructs | No. of Independent Transgenic Lines | Mean Percentage of GUS Negative T1 Seedlings | No. of Lines with 100% Excision Efficiency |
---|---|---|---|---|
Experiment No. 1 | Control Cassette | 22 | 19.2% | 0 |
Gene Deletor | 66 | 37.7% | 2 | |
Experiment No. 2 | Control Cassette | 19 | 21.4% | 0 |
Gene Deletor | 81 | 36.1% | 2 | |
Experiment No. 3 | Control Cassette | 21 | 20.7% | 0 |
Gene Deletor | 55 | 35.0% | 1 |
Line | Transgene Copies | Self-Pollinated | WT† as Pollen Recipient | WT as Pollen Donor | Transgene Excision Efficiency (%) | |||
---|---|---|---|---|---|---|---|---|
GUS− | GUS+ | GUS− | GUS+ | GUS− | GUS+ | |||
C1 | 1 | 532 | 1742 | 789 | 834 | 873 | 951 | 0.0 |
D4 | 1 | 21,862 | 0 | 2654 | 0 | 2476 | 0 | 100.0 |
D10 | 1 | 41,787 | 0 | 3844 | 0 | 4366 | 0 | 100.0 |
C14 | 1 | 651 | 1938 | 745 | 817 | 922 | 893 | 0.0 |
D31 | 1 | 6874 | 0 | 2943 | 0 | 3326 | 0 | 100.0 |
D56 | 1 | 5796 | 0 | 3219 | 0 | 3543 | 0 | 100.0 |
C6 | 1 | 717 | 2258 | 1142 | 1271 | 967 | 1055 | 0.0 |
D43 | 1 | 5864 | 0 | 2227 | 0 | 2581 | 0 | 100.0 |
Experiment No. | Number of Field Tests | Line | Self-Pollinated | WT as Pollen Recipient | WT as Pollen Donor | Transgene Excision Efficiency (%) | |||
---|---|---|---|---|---|---|---|---|---|
GUS− | GUS+ | GUS− | GUS+ | GUS− | GUS+ | ||||
Experiment 1 | Field Test (1) | C1 | 658 | 2046 | 892 | 973 | 795 | 870 | 0.0 |
D4 | 49,264 | 0 | 6739 | 0 | 6025 | 0 | 100.0 | ||
D10 | 53,119 | 0 | 6033 | 0 | 6989 | 0 | 100.0 | ||
Field Test (2) | C 1 | 506 | 1647 | 768 | 794 | 691 | 715 | 0.0 | |
D4 | 44,301 | 0 | 5541 | 0 | 5905 | 0 | 100.0 | ||
D10 | 64,356 | 0 | 7647 | 0 | 8036 | 0 | 100.0 | ||
Field Test (3) | C1 | 1628 | 5034 | 1773 | 1826 | 1698 | 1735 | 0.0 | |
D4 | 20,449 | 0 | 4363 | 0 | 4834 | 0 | 100.0 | ||
D10 | 16,841 | 0 | 3961 | 0 | 3589 | 0 | 100.0 | ||
Experiment 2 | Field Test (4) | C14 | 1275 | 3922 | 1169 | 1352 | 1288 | 1346 | 0.0 |
D31 | 18,864 | 0 | 4032 | 0 | 3975 | 0 | 100.0 | ||
D56 | 10,011 | 0 | 2763 | 0 | 2970 | 0 | 100.0 | ||
Field Test (5) | C14 | 1053 | 3217 | 976 | 1018 | 1127 | 1253 | 0.0 | |
D31 | 16,797 | 0 | 3524 | 0 | 3276 | 0 | 100.0 | ||
D56 | 16,231 | 0 | 3226 | 0 | 3537 | 0 | 100.0 | ||
Experiment 3 | Field Test (6) | C6 | 1136 | 3579 | 1217 | 1188 | 1204 | 1256 | 0.0 |
D43 | 14,683 | 0 | 2533 | 0 | 2369 | 0 | 100.0 | ||
Field Test (7) | C6 | 1317 | 4183 | 1479 | 1514 | 1396 | 1447 | 0.0 | |
D43 | 16,068 | 0 | 2699 | 0 | 2528 | 0 | 100.0 |
Line | PCR Analysis of Self-Pollinated T1 Seedlings | DNA Sequencing Analysis of 0.2 kb Fragments in T1 Seedlings | ||||
---|---|---|---|---|---|---|
Seedlings Carrying the 7.6 kb Fragment | Seedlings Carrying the 0.2 kb Fragment | Seedlings Not Producing Any Bands | Total Seedlings Tested for PCR | Seedlings Sequenced | Seedlings Carrying Expected 0.2 kb Sequence | |
C1 | 21 | 0 | 9 | 30 | 0 | 0 |
D4 | 0 | 38 | 12 | 50 | 10 | 10 |
D10 | 0 | 36 | 14 | 50 | 10 | 10 |
D31 | 0 | 33 | 17 | 50 | 5 | 5 |
D56 | 0 | 37 | 13 | 50 | 5 | 5 |
D43 | 0 | 40 | 10 | 50 | 5 | 5 |
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Duan, Z.; He, M.; Akbar, S.; Zhao, D.; Zhang, M.; Li, Y.; Yao, W. Confirmation of ‘Pollen- and Seed-Specific Gene Deletor’ System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions. Int. J. Mol. Sci. 2023, 24, 1160. https://doi.org/10.3390/ijms24021160
Duan Z, He M, Akbar S, Zhao D, Zhang M, Li Y, Yao W. Confirmation of ‘Pollen- and Seed-Specific Gene Deletor’ System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions. International Journal of Molecular Sciences. 2023; 24(2):1160. https://doi.org/10.3390/ijms24021160
Chicago/Turabian StyleDuan, Zhenzhen, Mingyang He, Sehrish Akbar, Degang Zhao, Muqing Zhang, Yi Li, and Wei Yao. 2023. "Confirmation of ‘Pollen- and Seed-Specific Gene Deletor’ System Efficiency for Transgene Excision from Transgenic Nicotiana tabacum under Field Conditions" International Journal of Molecular Sciences 24, no. 2: 1160. https://doi.org/10.3390/ijms24021160