利用Bulk-Way转化法获得甘蓝型油菜BBM-GR转化植株
1. 辽宁省农业科学院, 高粱研究所, 辽宁沈阳 110161
2. 沈阳药科大学, 功能食品与葡萄酒学院, 辽宁沈阳 110016
| 摘 要： | BBM（Baby-boom）是重要的植物胚胎发生相关转录因子，近年来在转基因顽抗种受体再生和植物体细胞胚胎发生（somatic embryogenesis）研究中起到关键作用。但目前在芸薹属作物中，BBM转录因子的剂量效应和时间窗口仍然未知。糖皮质激素受体系统（glucocorticoid receptor system，GR）和地塞米松（dexamethasone）是定时定量分析转录因子的重要研究工具。本研究首先利用BP、LR反应，构建了czn3999 35S::omega-BBM-GR载体；通过电击转化法将czn3999 35S::omega-BBM-GR质粒转入大肠杆菌DH5α及农杆菌C58的感受态细胞中，将PCR酶切验证的菌株制成甘油菌，-80°C备用。再利用农杆菌C58菌株介导的bulk-way转化技术，以甘蓝型油菜代表性品种12075子叶柄为转化受体，共培养后诱导地上部生长，生根处理后获得可育转基因阳性植株22株。本研究详尽梳理了Bulk-Way转化法的实验流程，为分析BBM的剂量效应及时间窗口提供了宝贵的研究材料。 |
| 关 键 词： | 转录因子; BBM; 糖皮质激素受体系统; 芸薹属; 遗传转化 |
| DOI： | 10.57237/j.life.2023.02.003 |
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收稿日期: 2023-03-28
接受日期: 2023-05-23
在线出版日期: 2023-06-15
Brassica napus BBM-GR Transformants from Bulk-Way Transformation
1. Sorghum Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang 110161, China
2. Faculty of Functional Food and Wine,, Shenyang Pharmaceutical University, Shenyang 110016, China
| Abstract: | BBM (baby boom) is an important embryogenesis transcription factor in plants, which is valuable in molecular research and could improve the transformation efficiency in recalcitrant species. However, in time and dose aspect, which stage and how much BBM transcription factor is necessary for stimulating embryogenesis is still largely unknown in Brassicas. GR system and dexamethasone are important to timing and quantitative analysis of transcription factors. In this study, BP and LR reaction were applied to build czn3999-35s: Omega-BBM-GR construct, which was transformed into E. coli DH5 α and Agrobacterium C58 competent cells by electric shock transformation respectively. The correct strain verified by PCR digestion reaction was kept in glycerol and stored at -80°C. Brassica napus 12075 cotyledon petioles were used as recipient, and eventually 22 transgenic plantlets were obtained from agrobacterium mediated bulk-way transformation. We describe Bulk-Way transformation protocol in detail, and provides valuable materials for analyzing dose and time effect of BBM in Brassicas. |
| Keywords: | Transcription Factor; BBM; Glucocorticoid Receptor System; Brassicas; Genetic Transformation |
基金
| 1. | 中国博士后科学基金面上项目 (2021M693846) |
参考文献
| [1] | Aregawi, K., Shen, J., Pierroz, G., Sharma, M. K., Dahlberg, J., Owiti, J., & Lemaux, P. G. Morphogene-assisted transformation of Sorghum bicolor allows more efficient genome editing. Plant Biotechnology Journal, (2022). 20 (4), 748–760. https://doi.org/10.1111/pbi.13754 |
| [2] | Boutilier, K., Offringa, R., Sharma, V. K., Kieft, H., Ouellet, T., Zhang, L., Hattori, J., Liu, C. M., Van Lammeren, A. A. M., Miki, B. L. A., Custers, J. B. M., & Van Lookeren Campagne, M. M.. Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. Plant Cell, (2002) 14 (8), 1737–1749. https://doi.org/10.1105/tpc.001941 |
| [3] | Deng, W., Luo, K., Li, Z., & Yang, Y. Plant Science A novel method for induction of plant regeneration via somatic embryogenesis. Plant Science (2009). 177, 43–48. https://doi.org/10.1016/j.plantsci.2009.03.009 |
| [4] | Florez, S. L., Erwin, R. L., Maximova, S. N., Guiltinan, M. J., & Curtis, W. R.. Enhanced somatic embryogenesis in Theobroma cacao using the homologous BABY BOOM transcription factor. BMC Plant Biology, (2015) 15 (1). https://doi.org/10.1186/s12870-015-0479-4 |
| [5] | Heidmann, I., de Lange, B., Lambalk, J., Angenent, G. C., & Boutilier, K. Efficient sweet pepper transformation mediated by the BABY BOOM transcription factor. Plant Cell Reports, (2011). 30 (6), 1107–1115. https://doi.org/10.1007/s00299-011-1018-x |
| [6] | Horstman, A., Bemer, M., & Boutilier, K.. A transcriptional view on somatic embryogenesis. Regeneration, (2017) 4 (4), 201–216. https://doi.org/10.1002/reg2.91 |
| [7] | Horstman, A., Fukuoka, H., Muino, J. M., Nitsch, L., Guo, C., & Passarinho, P. AIL and HDG proteins act antagonistically to control cell proliferation STEM CELLS AND REGENERATION AIL and HDG proteins act antagonistically to control cell proliferation. January. (2015). https://doi.org/10.1242/dev.117168 |
| [8] | Li, H., Soriano, M., Cordewener, J., Muiño, J. M., Riksen, T., Fukuok, H., Angenent, G. C., & Boutilier, K. The histone deacetylase inhibitor trichostatin a promotes totipotency in the male gametophyte. Plant Cell, (2014). 26 (1), 195–209. https://doi.org/10.1105/tpc.113.116491 |
| [9] | Lowe, K., Wu, E., Wang, N., Hoerster, G., Hastings, C., Cho, M., Scelonge, C., Lenderts, B., Chamberlin, M., Cushatt, J., Wang, L., Ryan, L., Khan, T., Chow-yiu, J., Hua, W., Yu, M., Banh, J., Bao, Z., Brink, K., … Marredpally, W. Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation. (2016). https://doi.org/10.1105/tpc.16.00124 |
| [10] | Lutz, K. A., Azhagiri, A., & Maliga, P. Transplastomics in arabidopsis: Progress toward developing an efficient method. Methods in Molecular Biology, (2011). 774 (7), 133–147. https://doi.org/10.1007/978-1-61779-234-2_9 |
| [11] | Maher, M. F., Nasti, R. A., Vollbrecht, M., Starker, C. G., Clark, M. D., & Voytas, D. F. Plant gene editing through de novo induction of meristems. Nature Biotechnology, (2020). 38 (1), 84–89. https://doi.org/10.1038/s41587-019-0337-2 |
| [12] | Moore, I., Samalova, M., & Kurup, S. Transactivated and chemically inducible gene expression in plants. Plant Journal, (2006). 45 (4), 651–683. https://doi.org/10.1111/j.1365-313X.2006.02660.x |
| [13] | Passarinho, P., Ketelaar, T., Xing, M., Van Arkel, J., Maliepaard, C., Hendriks, M. W., Joosen, R., Lammers, M., Herdies, L., Den Boer, B., Van Der Geest, L., & Boutilier, K. BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways. Plant Molecular Biology, (2008). 68 (3), 225–237. https://doi.org/10.1007/s11103-008-9364-y |
| [14] | Zhang, X., Xu, G., Cheng, C., Lei, L., Sun, J., Xu, Y., Deng, C., Dai, Z., Yang, Z., Chen, X., Liu, C., Tang, Q., & Su, J. Establishment of an Agrobacterium-mediated genetic transformation and CRISPR/Cas9-mediated targeted mutagenesis in Hemp (Cannabis Sativa L.). Plant Biotechnology Journal, (2021). 19 (10), 1979–1987. https://doi.org/10.1111/pbi.13611 |
| [15] | 罗文新, 张军, 杨海杰, 李少伟, 谢小燕, 逄淑强, 李少菁, 夏宁邵. 一种带增强子的原核高效表达载体的构建及初步应用. 生物工程学报, (2000). 16 (5), 3–6. |
