农业细菌-中生菌遗传转化、转基因生产及其在水稻雄性生殖发育研究中的应用
1Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University-University of Adelaide Joint Centre for Agriculture and Health, School of Life Sciences and Biotechnology,Shanghai Jiao Tong University, 2College of Agronomy,Anhui Agricultural University, Heifei, China, 3School of Agriculture, Food and Wine,University of Adelaide
Summary
这项工作描述了使用CRISPR-Cas9基因组编辑技术敲除内源 基因OsABCG15， 然后修改的农业细菌 中培养转化方案，在水稻中产生稳定的雄性-无菌线。
Abstract
雄性不育是杂交种子生产的重要农艺特征，通常以雄性生殖器官/游戏体的功能缺陷为特征。CRISPR-Cas9基因组编辑技术的最新进展使特定位点内源候选基因的编辑效率高，而且可以节省时间。此外， 水稻的农业细菌中位基因转化也是基因改造的关键方法，已被许多公立和私营实验室广泛采用。在这项研究中，我们应用CRISPR-Cas9基因组编辑工具，通过目标基因组编辑 OsABCG15在japonica 培养中成功生成三 条雄性无菌 突变线。我们采用一 种改良的农业细菌调剂水稻转化方法，为水稻杂交种子生产提供了优良的遗传消瘦手段。转基因植物可以在2~3个月内获得，通过PCR扩增和桑格测序通过基因分型筛选同源转化剂。通过对水稻雄性生殖器官的微观观察，通过碘碘化钾（I2-KI）染色半薄横截面进行花粉生存能力分析，对雄性无菌同源线进行基本表型表征。
Introduction
稻米是最重要的粮食作物，特别是在发展中国家，是世界人口一半以上的主食。总体而言，对稻谷的需求正在增长，预计到2030年将增长50%，到2050年将增长100%。,2未来水稻产量的提高需要利用多样化的分子和遗传资源，使水稻成为单体植物研究的优秀模型。其中包括一个有效的转换系统，先进的分子图，和公开访问的数据库的表达序列标签，这是多年来产生的,3，4。提高作物产量的一个策略是杂交种子生产5，其中一个核心要素是操纵男性生育能力。了解谷物作物中男性肥力的分子控制有助于将关键知识转化为实用技术，以提高杂交种子产量，提高作物,产量。
基因转化是基础研究和商业农业的关键工具，因为它能够引进外来基因或操纵作物中的内源基因，并产生转基因线。适当的转化方案可以帮助加速遗传和分子生物学研究，以基本理解基因调控8。在细菌中，遗传转化是自然发生的;然而，在植物中，它是使用分子生物学技术9，10,人为执行的。农业细菌Tumefaciens是一种土壤传播的、格拉姆阴性细菌，通过T-DNA（Ti质粒的一个区域）通过IV型分泌系统11，12将T-DNA转移到植物细胞中，导致植物冠胆病。在植物中，A.tumefaciens-中照转化被认为是一种广泛的基因修饰方法，因为它导致T-DNA稳定且低拷贝数集成到宿主基因组13中。转基因水稻是1990Agrobacterium年代中期在雅波尼卡品种14中首次通过农业细菌培养基因转化产生的。利用该协议，在4个月内获得多条转基因线路，转化效率为10%~30%。研究表明，成功转化有两个关键步骤：一是胚胎基因从成熟种子中诱导，另一个是将乙酰胺酮（一种酚类化合物）加入到共同培养的细菌培养中，从而在14、15,植物中提高转化效率。该协议已广泛使用与小改动在japonica16，,17，,18， 19,19以及其他品种，如稻谷20， 21，,21,22，,23和热带贾波尼卡24，,25.事实上，超过80%的关于水稻转化的文章使用农业细菌中位基因转化作为工具13。迄今为止，已经开发出几种基因转化方案，利用水稻种子作为卡卢斯诱导16、17、18、19,17,18,的起始材料。然而，对于年轻的花序作为卡卢斯生产的外植，却很少为人所知。总体而言，为功能基因组学和作物改良研究建立快速、可重复、高效的基因转化和再生协议非常重要。
近年来，CRISPR-Cas9技术的发展产生了精确的基因组编辑机制，以了解基因功能，为植物育种提供农,27艺上的重要改进。CRISPR也为男性生殖发育和杂交生产提供了相当大的希望。在这项研究中，我们利用CRISPR-Cas9技术利用基因敲除系统，并结合使用幼年花序作为外植的高效水稻基因转化方案，从而为研究生殖发育创造稳定的雄性无菌线。
Protocol
1. sgRNA-CAS9植物表达载体 构造和农业细菌-中栽转化 根据28日出版的文献，在水稻中瞄准雄性无菌基因OsABCG15。 在 OsABCGG15 的第二个 exon 中，为位于 106-125 bp 之间的目标站点设计 sgRNA（图 1）。 使用 T4 多核苷酸激酶合成 sgRNA 寡糖 （sgR-osABCG15-F： 5’TGGCAAGCACATCCTCAAGAT3′ 和 5’sgR-OsABCG15-R：ACATCCCCTGTGTGTT’）。 使用内核…
Representative Results
这里演示的是利用基因编辑技术，为水稻中农业细菌的遗传转化的未来研究创建一条雄性无菌线。为了创建雄性无菌线osabcg15，CRISPR-CAS9中位突变用于二元向量构造。sgRNA由OsU3启动子驱动，而hSpCas9的表达盒是由双35S启动子驱动的，中间载体组装在一个二进制载体pCAMBIA1300中，专为农业细菌中调解的水稻稳定转化而设计。图1A提供了用于CRISPR/Cas9中切水稻突变的构造的<strong …
Discussion
人工原生雄性无菌突变体传统上由随机的物理、化学或生物突变产生。虽然这些都是强大的技术，他们的随机性质未能利用大量的现代基因组知识，有可能提供定制的改进分子育种32。CRISPR-Cas9系统由于其操作和编辑DNA29，33的简单而经济实惠的手段，在植物中得到了广泛的应用。与传统的诱变方法相比，它具有节省时间的潜力。
<p c…Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢陈小飞为幼米提供花序，协助制作水稻组织培养媒介。这项工作得到了国家自然科学基金（31900611）的支持。
Materials
| 1-Naphthaleneacetic acid | Sigma-Aldrich | N0640 | |
| 2，4-Dichlorophenoxyacetic Acid | Sigma-Aldrich | D7299 | |
| 6-Benzylaminopurine (6-BA) | Sigma-Aldrich | B3408 | |
| Acetosyringone | Sigma-Aldrich | D134406 | |
| Agar | Sinopharm Chemical Reagent Co., Ltd | 10000561 | |
| Ammonium sulfate | Sinopharm Chemical Reagent Co., Ltd | 10002918 | |
| Aneurine hydrochloride | Sigma-Aldrich | T4625 | |
| Anhydrous ethanol | Sinopharm Chemical Reagent Co., Ltd | 10009218 | |
| Bacteriological peptone | Sangon Biotech | A100636 | |
| Beef extract | Sangon Biotech | A600114 | |
| Boric acid | Sinopharm Chemical Reagent Co., Ltd | 10004808 | |
| Calcium chloride dihydrate | Sinopharm Chemical Reagent Co., Ltd | 20011160 | |
| Casein acid hydrolysate | Beijing XMJ Scientific Co., Ltd | C184 | |
| Cobalt(Ⅱ) chloride hexahydrate | Sinopharm Chemical Reagent Co., Ltd | 10007216 | |
| Copper(Ⅱ) sulfate pentahydrate | Sinopharm Chemical Reagent Co., Ltd | 10008218 | |
| D(+)-Glucose anhydrous | Sinopharm Chemical Reagent Co., Ltd | 63005518 | |
| D-sorbitol | Sinopharm Chemical Reagent Co., Ltd | 63011037 | |
| EDTA, Disodium Salt, Dihydrate | Sigma-Aldrich | E5134 | |
| EOS Digital SLR and Compact System Cameras | Canon | EOS 700D | |
| Formaldehyde | Sinopharm Chemical Reagent Co., Ltd | 10010018 | |
| Fully Automated Rotary Microtome | Leica Biosystems | Leica RM 2265 | |
| Glacial acetic acid | Sinopharm Chemical Reagent Co., Ltd | 10000208 | |
| Glycine | Sinopharm Chemical Reagent Co., Ltd | 62011516 | |
| Hygromycin | Beijing XMJ Scientific Co., Ltd | H370 | |
| Inositol | Sinopharm Chemical Reagent Co., Ltd | 63007738 | |
| Iodine | Sinopharm Chemical Reagent Co., Ltd | 10011517 | |
| Iron(Ⅱ) sulfate heptahydrate | Sinopharm Chemical Reagent Co., Ltd | 10012116 | |
| Kanamycine | Beijing XMJ Scientific Co., Ltd | K378 | |
| Kinetin | Sigma-Aldrich | K0753 | |
| L-Arginine | Sinopharm Chemical Reagent Co., Ltd | 62004034 | |
| L-Aspartic acid | Sinopharm Chemical Reagent Co., Ltd | 62004736 | |
| L-Glutamine | Beijing XMJ Scientific Co., Ltd | G229 | |
| L-proline | Beijing XMJ Scientific Co., Ltd | P698 | |
| Magnesium sulfate heptahydrate | Sinopharm Chemical Reagent Co., Ltd | 10013018 | |
| Manganese sulfate monohydrate | Sinopharm Chemical Reagent Co., Ltd | 10013418 | |
| Microscopes | NIKON | Eclipse 80i | |
| MS | Phytotech | M519 | |
| Nicotinic acid | Sigma-Aldrich | N0765 | |
| Phytagel | Sigma-Aldrich | P8169 | |
| Potassium chloride | Sinopharm Chemical Reagent Co., Ltd | 10016308 | |
| Potassium dihydrogen phosphate | Sinopharm Chemical Reagent Co., Ltd | 10017608 | |
| Potassium iodide | Sinopharm Chemical Reagent Co., Ltd | 10017160 | |
| Potassium nitrate | Sinopharm Chemical Reagent Co., Ltd | 1001721933 | |
| Pyridoxine Hydrochloride (B6) | Sigma-Aldrich | 47862 | |
| Rifampicin | Beijing XMJ Scientific Co., Ltd | R501 | |
| Sodium hydroxide | Sinopharm Chemical Reagent Co., Ltd | 10019718 | |
| Sodium molybdate dihydrate | Sinopharm Chemical Reagent Co., Ltd | 10019816 | |
| Stereo microscopes | Leica Microsystems | Leica M205 A | |
| Sucrose | Sinopharm Chemical Reagent Co., Ltd | 10021418 | |
| Technovit embedding Kits 7100 | Heraeus Teknovi, Germany | 14653 | |
| Timentin | Beijing XMJ Scientific Co., Ltd | T869 | |
| Toluidine Blue O | Sigma-Aldrich | T3260 | |
| Water bath for paraffin sections | Leica Biosystems | Leica HI1210 | |
| Yeast extract | Sangon Biotech | A515245 | |
| Zinc sulfate heptahydrate | Sinopharm Chemical Reagent Co., Ltd | 10024018 |
References
- Izawa, T., Shimamoto, K. Becoming a model plant: The importance of rice to plant science. Trends in Plant Science. 1 (3), 95-99 (1996).
- Shimamoto, K., Kyozuka, J. Rice as a model for comparative genomics of plants. Annual Review of Plant Biology. 53 (1), 399-419 (2002).
- Selva, C., et al. Hybrid breeding in wheat: how shaping floral biology can offer new perspectives. Functional Plant Biology. 47 (8), 675-694 (2020).
- Lippman, Z. B., Zamir, D. Heterosis: revisiting the magic. Trends in Genetics. 23 (2), 60-66 (2007).
- Zhang, D., Liang, W. Improving food security: using male fertility for hybrid seed breeding. Science. , 45-48 (2016).
- Masters, A., et al. Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes. Journal of Visualized Experiments. (156), e60782 (2020).
- Laurenceau, R., et al. A type IV pilus mediates DNA binding during natural transformation in Streptococcus pneumoniae. PLoS Pathogens. 9 (6), 1003473 (2013).
- Tzfira, T., Citovsky, V. Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Current Opinion in Biotechnology. 17 (2), 147-154 (2006).
- Gelvin, S. B. Agrobacterium in the genomics age. Plant Physiology. 150 (4), 1665-1676 (2009).
- Lacroix, B., Citovsky, V. The roles of bacterial and host plant factors in Agrobacterium-mediated genetic transformation. International Journal of Developmental Biology. 57, 467-481 (2013).
- Hiei, Y., Komari, T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nature Protocols. 3 (5), 824 (2008).
- Hiei, Y., Ohta, S., Komari, T., Kumashiro, T. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal. 6 (2), 271-282 (1994).
- Hiei, Y., Komari, T., Kubo, T. Transformation of rice mediated by Agrobacterium tumefaciens. Plant Molecular Biology. 35 (1-2), 205-218 (1997).
- Nishimura, A., Aichi, I., Matsuoka, M. A protocol for Agrobacterium-mediated transformation in rice. Nature Protocols. 1 (6), 2796 (2006).
- Yara, A., et al. Production of transgenic japonica rice (Oryza sativa) cultivar, Taichung 65, by the Agrobacterium-mediated method. Plant Biotechnology. 18 (4), 305-310 (2001).
- Cho, S. K., et al. Efficient transformation of Korean rice cultivars (Oryza sativa L.) mediated by Agrobacterium tumefaciens. Journal of Plant Biology. 41 (4), 262-268 (1998).
- Toki, S. Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Molecular Biology Reporter. 15, 16-21 (1997).
- Zhang, J., Xu, R. j., Elliott, M. C., Chen, D. F. Agrobacterium-mediated transformation of elite indica and japonica rice cultivars. Molecular Biotechnology. 8 (3), 223-231 (1997).
- Aldemita, R. R., Hodges, T. K. Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties. Planta. 199 (4), 612-617 (1996).
- Rashid, H., Yokoi, S., Toriyama, K., Hinata, K. Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Reports. 15 (10), 727-730 (1996).
- Sahoo, K. K., Tripathi, A. K., Pareek, A., Sopory, S. K., Singla-Pareek, S. L. An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods. 7 (1), 49 (2011).
- Rachmawati, D., Hosaka, T., Inoue, E., Anzai, H. Agrobacterium-mediated transformation of Javanica rice cv. Rojolele. Bioscience, Biotechnology, and Biochemistry. 68 (6), 1193-1200 (2004).
- Dong, J., Teng, W., Buchholz, W. G., Hall, T. C. Agrobacterium-mediated transformation of Javanica rice. Molecular Breeding. 2 (3), 267-276 (1996).
- Bortesi, L., Fischer, R. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances. 33 (1), 41-52 (2015).
- Li, Q., et al. Development of japonica photo-sensitive genic male sterile rice lines by editing carbon starved anther using CRISPR/Cas9. Journal of Genetics and Genomics. 43 (6), 415 (2016).
- Qin, P., et al. ABCG15 encodes an ABC transporter protein, and is essential for Post-Meiotic anther and pollen exine development in rice. Plant and Cell Physiology. 54, (2013).
- Mao, Y., et al. Application of the CRISPR-Cas system for efficient genome engineering in plants. Molecular Plant. 6 (6), 2008-2011 (2013).
- Itoh, J. I., et al. Rice plant development: from zygote to spikelet. Plant and Cell Physiology. 46 (1), 23-47 (2005).
- Gawel, N. J., Jarret, R. L. A modified CTAB DNA extraction procedure forMusa andIpomoea. Plant Molecular Biology Reporter. 9 (3), 262-266 (1991).
- Wei, F. J., Droc, G., Guiderdoni, E., Hsing, Y. i. C. International Consortium of Rice Mutagenesis: resources and beyond. Rice. 6 (1), 39 (2013).
- Feng, Z., et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell Research. 23 (10), 1229 (2013).
Cite This Article
Xu, D., Mondol, P. C., Uzair, M., Tucker, M. R., Zhang, D. Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice. J. Vis. Exp. (164), e61665, doi:10.3791/61665 (2020).