Variations of Alkaloid Accumulation and Gene Transcription in Nicotiana tabacum
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. Alkaloid Analysis
2.3. Quantitative Real Time PCR Analysis
2.4. Statistical Analysis
3. Results and Discussion
3.1. The Composition and Content of Alkaloids
3.2. The Transcription Levels of the Key Structural Genes of Alkaloid Biosynthesis
3.3. Variance Analysis of Genetic Effects
3.4. The Correlation among Different Alkaloids
4. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Variety | Site | Developmental Stage | Sampling Leaf Position | Sampling Time (yy/mm/dd) |
---|---|---|---|---|
Hongda | Xiangyun county, Yunnan Province, China | Rosette stage | Middle part | 11/06/20 |
Vigorous growing stage | Middle part | 11/07/06 | ||
Lower leaves maturing stage | 4–6 | 11/07/23 | ||
Middle leaves maturing stage | 11–13 | 11/08/09 | ||
Upper leaves maturing stage | 16–18 | 11/08/25 | ||
K326 | Zunyi county, Guizhou Province, China | Rosette stage | Middle part | 11/06/20 |
Vigorous growing stage | Middle part | 11/07/06 | ||
Lower leaves maturing stage | 4–6 | 11/07/23 | ||
Middle leaves maturing stage | 11–13 | 11/08/09 | ||
Upper leaves maturing stage | 16–18 | 11/08/25 | ||
Zhongyan 100 | Xiangxian county, Henan Province, China | Rosette stage | Middle part | 11/06/20 |
Vigorous growing stage | Middle part | 11/07/06 | ||
Lower leaves maturing stage | 4–6 | 11/07/23 | ||
Middle leaves maturing stage | 11–13 | 11/08/09 | ||
Upper leaves maturing stage | 16–18 | 11/08/25 |
Gene Name | Forward (F) or Reverse (R) | Sequence | Reference |
---|---|---|---|
ODC | F | ACTGTGTTTGGGCCCACTTG | [4] |
R | CCATATTAGGAAAAACCAGC | ||
PMT | F | ATTGGACCAAGATCGAGTC | [4] |
R | ATTACTGCAGAATTCTCCTAC | ||
MPO | F | CAGTGATGTTACTGAAACTA | [4] |
R | ATAGGCGAGGAGGACTCATG | ||
AO | F | TTAACAAAGTCATCCGTCGG | [4] |
R | ATTTAGTCTTGAGGTAGACC | ||
QS | F | AATCACTGCTTGATGGTATC | [4] |
R | ACTGGCAAGTTCTTGGACTC | ||
QPT | F | GACGCATTCCGTGAAAGCAC | [4] |
R | AAGTAATGGCGCTCATGCTC | ||
26S-RNA | F | GAAGAAGGTCCCAAGGGTTC | [16] |
R | TCTCCCTTTAACACCAACGG |
Variety | Developmental Stage | Nicotine (mg g−1 DW) | Nornicotine (μg g−1 DW) | Myosmine (μg g−1 DW) | Anabasine (μg g−1 DW) | Anatabine (μg g−1 DW) | Cotinine (μg g−1 DW) | Total Alkaloids (mg g−1 DW) |
---|---|---|---|---|---|---|---|---|
Hongda | Rosette stage | 7.31 ± 0.35 g | 47.12 ± 2.55 f | 3.95 ± 0.48 f | 6.21 ± 0.73 g | 97.38 ± 9.06 h | 5.96 ± 1.25 e | 7.47 ± 0.36 g |
Vigorous growing stage | 6.22 ± 0.49 g | 37.77 ± 4.07 f | 3.39 ± 0.58 f | 8.53 ± 1.25 g | 180.58 ± 32.41 gh | 4.58 ± 0.40 e | 6.46 ± 0.52 g | |
Lower leaves maturing stage | 7.59 ± 0.66 g | 54.03 ± 0.94 f | 3.70 ± 0.06 f | 10.48 ± 1.00 g | 286.95 ± 45.29 g | 6.15 ± 1.04 e | 7.95 ± 0.66 g | |
Middle leaves maturing stage | 11.70 ± 0.22 f | 124.77 ± 23.88 e | 12.22 ± 1.54 de | 27.75 ± 2.17 f | 607.87 ± 60.02 f | 34.39 ± 2.54 c | 12.50 ± 0.24 f | |
Upper leaves maturing stage | 21.85 ± 2.20 cd | 321.10 ± 11.40 cd | 17.10 ± 3.22 d | 76.72 ± 7.22 c | 1307.54 ± 126.89 c | 8.25 ± 0.49 e | 23.58 ± 2.32 cd | |
K326 | Rosette stage | 5.77 ± 0.26 g | 47.52 ± 2.65 f | 4.04 ± 1.69 f | 5.63 ± 0.19 g | 104.41 ± 8.28 h | 5.71 ± 1.95 e | 5.94 ± 0.26 g |
Vigorous growing stage | 11.34 ± 1.06 f | 119.31 ± 20.41 e | 9.38 ± 1.64 e | 13.35 ± 0.17 g | 273.91 ± 39.38 g | 7.71 ± 1.21 e | 11.76 ± 1.10 f | |
Lower leaves maturing stage | 22.97 ± 1.69 c | 347.05 ± 34.48 c | 27.54 ± 2.90 c | 33.99 ± 2.69 ef | 738.93 ± 38.97 ef | 33.05 ± 5.25 c | 24.15 ± 1.69 c | |
Middle leaves maturing stage | 20.75 ± 0.73 d | 358.34 ± 16.00 c | 36.42 ± 5.64 b | 49.28 ± 5.06 d | 933.56 ± 187.86 d | 72.70 ± 12.28 a | 22.20 ± 0.67 cd | |
Upper leaves maturing stage | 38.82 ± 0.51 a | 1055.81 ± 94.16 a | 58.41 ± 3.52 a | 128.18 ± 8.66 a | 2029.32 ± 179.12 a | 43.67 ± 2.29 b | 42.13 ± 0.72 a | |
Zhongyan 100 | Rosette stage | 7.25 ± 0.12 g | 49.03 ± 3.81 f | 3.82 ± 0.42 f | 6.87 ± 0.85 g | 102.53 ± 8.67 h | 6.28 ± 1.17 e | 7.42 ± 0.12 g |
Vigorous growing stage | 5.73 ± 0.68 g | 46.02 ± 2.48 f | 2.46 ± 0.22 f | 6.67 ± 0.94 g | 133.08 ± 14.44 h | 2.45 ± 0.20 e | 5.92 ± 0.69 g | |
Lower leaves maturing stage | 18.11 ± 0.59 e | 280.40 ± 52.63 d | 16.43 ± 0.97 d | 33.27 ± 1.38 ef | 702.27 ± 34.30 f | 19.01 ± 2.94 d | 19.16 ± 0.67 e | |
Middle leaves maturing stage | 20.29 ± 1.13 d | 369.45 ± 15.49 c | 23.11 ± 1.52 c | 42.67 ± 3.35 de | 873.33 ± 60.32 de | 42.32 ± 9.34 b | 21.65 ± 1.19 d | |
Upper leaves maturing stage | 27.38 ± 3.12 b | 897.02 ± 28.12 b | 27.49 ± 8.15 c | 104.20 ± 21.69 b | 1881.15 ± 67.92 b | 25.55 ± 5.22 d | 30.32 ± 3.17 b |
Trait | Developmental Stage | ODC | PMT | MPO | AO | QS | QPT |
---|---|---|---|---|---|---|---|
Vigorous Growing Stage | |||||||
Nicotine | Vigorous growing stage | 0.991 | 0.993 | 0.992 | 0.998 | 0.994 | 0.996 |
Nornicotine | 0.999 | 0.999 | 0.999 | 0.995 | 0.998 | 0.997 | |
Myosmine | 0.984 | 0.986 | 0.986 | 0.994 | 0.988 | 0.991 | |
Anabasine | 0.948 | 0.951 | 0.95 | 0.966 | 0.954 | 0.959 | |
Anatabine | 0.925 | 0.929 | 0.928 | 0.947 | 0.933 | 0.939 | |
Cotinine | 0.893 | 0.898 | 0.897 | 0.919 | 0.903 | 0.91 | |
Total alkaloids | 0.991 | 0.992 | 0.992 | 0.997 | 0.993 | 0.995 | |
Nicotine | Lower leaves maturing stage | 0.777 | 0.77 | 0.772 | 0.736 | 0.763 | 0.752 |
Nornicotine | 0.714 | 0.706 | 0.708 | 0.668 | 0.698 | 0.686 | |
Myosmine | 0.873 | 0.867 | 0.868 | 0.84 | 0.862 | 0.853 | |
Anabasine | 0.567 | 0.558 | 0.56 | 0.514 | 0.549 | 0.534 | |
Anatabine | 0.604 | 0.596 | 0.598 | 0.553 | 0.587 | 0.573 | |
Cotinine | 0.902 | 0.897 | 0.899 | 0.873 | 0.893 | 0.885 | |
Total alkaloids | 0.772 | 0.765 | 0.766 | 0.73 | 0.758 | 0.746 | |
Nicotine | Middle leaves maturing stage | 0.582 | 0.573 | 0.575 | 0.53 | 0.564 | 0.55 |
Nornicotine | 0.51 | 0.501 | 0.503 | 0.456 | 0.492 | 0.476 | |
Myosmine | 0.916 | 0.911 | 0.912 | 0.889 | 0.907 | 0.899 | |
Anabasine | 0.771 | 0.764 | 0.766 | 0.73 | 0.757 | 0.754 | |
Anatabine | 0.682 | 0.674 | 0.676 | 0.635 | 0.666 | 0.653 | |
Cotinine | 0.99 | 0.998 | 0.988 | 0.979 | 0.986 | 0.983 | |
Total alkaloids | 0.586 | 0.577 | 0.579 | 0.534 | 0.569 | 0.554 | |
Nicotine | Upper leaves maturing stage | 0.963 | 0.96 | 0.961 | 0.944 | 0.957 | 0.952 |
Nornicotine | 0.705 | 0.697 | 0.699 | 0.66 | 0.69 | 0.677 | |
Myosmine | 0.982 | 0.979 | 0.98 | 0.968 | 0.977 | 0.973 | |
Anabasine | 0.872 | 0.867 | 0.868 | 0.84 | 0.862 | 0.853 | |
Anatabine | 0.697 | 0.689 | 0.691 | 0.651 | 0.682 | 0.669 | |
Cotinine | 0.897 | 0.892 | 0.893 | 0.868 | 0.887 | 0.879 | |
Total alkaloids | 0.951 | 0.947 | 0.948 | 0.93 | 0.944 | 0.938 |
Parameter | Nicotine | Nornicotine | Myosmine | Anabasine | Anatabine | Cotinine | Total Alkaloids |
---|---|---|---|---|---|---|---|
VV/VP | 0.147 ** | 0.135 ** | 0.262 ** | 0.046 ** | 0.048 ** | 0.186 ** | 0.141 ** |
VD/VP | 0.683 ** | 0.655 ** | 0.468 ** | 0.865 ** | 0.887 ** | 0.611 ** | 0.700 ** |
VVD/VP | 0.153 ** | 0.204 ** | 0.231 ** | 0.053 ** | 0.050 ** | 0.170 ** | 0.144 ** |
Ve/VP | 0.018 | 0.006 | 0.038 * | 0.036 | 0.016 * | 0.033 ** | 0.016 |
Trait | Nicotine | Nornicotine | Myosmine | Anabasine | Anatabine | Cotinine | Total Alkaloids |
---|---|---|---|---|---|---|---|
Nicotine | 0.857 ** | 0.801 ** | 0.810 ** | 0.864 ** | 0.558 ** | 0.886 ** | |
0.878 ** | 0.838 ** | 0.827 ** | 0.881 ** | 0.583 ** | 0.898 ** | ||
Nornicotine | 0.900 ** | 0.775 ** | 0.843 ** | 0.895 ** | 0.483 ** | 0.868 ** | |
0.946 ** | 0.809 ** | 0.872 ** | 0.915 ** | 0.501 ** | 0.888 ** | ||
0.682 ** | |||||||
Myosmine | 0.832 ** | 0.809 ** | 0.698 ** | 0.750 ** | 0.657 ** | 0.807 ** | |
0.903 ** | 0.873 ** | 0.753 ** | 0.784 ** | 0.687 ** | 0.840 ** | ||
0.809 ** | 0.768 ** | ||||||
Anabasine | 0.818 ** | 0.851 ** | 0.768 ** | 0.877 ** | 0.422 ** | 0.821 ** | |
0.917 ** | 0.947 ** | 0.865 ** | 0.899 ** | 0.451 ** | 0.838 ** | ||
0.626 ** | 0.778 ** | 0.803 ** | |||||
Anatabine | 0.803 ** | 0.865 ** | 0.741 ** | 0.774 ** | 0.487 ** | 0.876 ** | |
0.973 ** | 0.981 ** | 0.939 ** | 0.954 ** | 0.509 ** | 0.892 ** | ||
0.666 ** | 0.861 ** | 0.709 ** | 0.663 ** | ||||
Cotinine | 0.851 ** | 0.817 ** | 0.854 ** | 0.780 ** | 0.734 ** | 0.557 ** | |
0.535 ** | 0.404 ** | 0.638 ** | 0.420 ** | 0.513 ** | 0.581 ** | ||
0.569 ** | 0.616 ** | 0.736 ** | 0.623 ** | 0.636 ** | |||
Total Alkaloids | 0.828 ** | 0.902 ** | 0.831 ** | 0.820 ** | 0.805 ** | 0.848 ** | |
0.947 ** | 0.950 ** | 0.906 ** | 0.922 ** | 0.977 ** | 0.532 ** | ||
0.848 ** | 0.712 ** | 0.819 ** | 0.644 ** | 0.688 ** | 0.585 ** |
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Sun, B.; Tian, Y.-X.; Zhang, F.; Chen, Q.; Zhang, Y.; Luo, Y.; Wang, X.-R.; Lin, F.-C.; Yang, J.; Tang, H.-R. Variations of Alkaloid Accumulation and Gene Transcription in Nicotiana tabacum. Biomolecules 2018, 8, 114. https://doi.org/10.3390/biom8040114
Sun B, Tian Y-X, Zhang F, Chen Q, Zhang Y, Luo Y, Wang X-R, Lin F-C, Yang J, Tang H-R. Variations of Alkaloid Accumulation and Gene Transcription in Nicotiana tabacum. Biomolecules. 2018; 8(4):114. https://doi.org/10.3390/biom8040114
Chicago/Turabian StyleSun, Bo, Yu-Xiao Tian, Fen Zhang, Qing Chen, Yong Zhang, Ya Luo, Xiao-Rong Wang, Fu-Cheng Lin, Jun Yang, and Hao-Ru Tang. 2018. "Variations of Alkaloid Accumulation and Gene Transcription in Nicotiana tabacum" Biomolecules 8, no. 4: 114. https://doi.org/10.3390/biom8040114