植物生长与农杆菌介导的植物浸水转化
Summary
利用花浸法进行农杆菌介导的转化, 可以成功地利用这种方法, 创造出稳定的极端植物模型schrenkiella parvula的转基因线。考虑到不同的生长习性和四肢的生理特性, 我们提出了一种从这种方法中修改的方案。
Abstract
夏氏叶炎是一种适应各种非生物胁迫的极端植物, 包括多重离子毒性胁迫。尽管有高质量的基因组资源可用于研究植物如何适应环境压力, 但由于缺乏可行的转化系统, 其作为功能基因组学模型和工具的价值受到限制。在这个协议中, 我们报告如何产生稳定的转基因s.类似线使用农杆菌介导的花浸法。我们修改了用于罗非鱼的转化协议, 以考虑到罗非鱼的独特特征, 如不确定的开花习惯和高表观蜡含量的叶子。简单地说, 在种植前,将其在4°c 下分层5天。植物生长在14小时的光和10小时黑暗的光周期和 130μmol m-2-1 的光强, 在 22°c 至24°c。选择了8至9个具有多个花序的周龄植物进行改造。这些花序被浸入携带 mp90rk质粒的农杆菌 gv3101 的浸润溶液中。我们在三到四周的时间间隔内进行了两轮插花, 以提高转化效率。t1 种子在萌发前在容器中收集并干燥四周, 以筛选出候选转化线。对 basta 的抗性被用来筛选 t1 植物。我们在两个为期一周的工厂喷洒了三次基本服务溶液, 间隔为三天, 以减少假阳性。对存活的单个植物进行了基本程度的空投试验, 以确定真正的阳性转化物。转化效率为 0.033, 每繁殖 10, 000 种 t1 种子产生3-4 个转基因植株。
Introduction
在这个协议中, 我们描述了生长和建立稳定的转基因线的极端植物模型雪伦基耶拉帕维拉。有效的转化系统的可用性是任何多功能遗传模型的标志。在极端环境中茁壮成长的植物, 被称为极端植物, 为了解植物对环境压力的适应提供了重要资源。施伦基耶拉·帕弗拉(原thellungiella parvula和eutrema parvula) 就是这样一种极端植物模型, 具有不断扩展的基因组资源1,2, 3,4, 5.然而, 在已发表的研究中, 还没有报道 s . parvula 的转化协议。
帕拉维的基因组是布拉西卡科 (芥末科) 首次公布的极生植物基因组, 并与非极生植物模型拟南芥1进行了广泛的整体基因组联合。因此,对塔利亚纳和阴道的比较研究可以受益于在塔利亚纳进行的大量基因研究, 以便对阴道基因组是如何进化和调控的做出信息性假设以应对极端环境压力5,6,7。在已知的野生亲缘植物中,黄花是最耐盐的物种之一 (基于土壤 ncl ld50)。除了氯化钠耐受性外, 在高浓度对大多数植物有毒的情况下, s. parvula还存活并完成其生命周期.为了应对其自然栖息地普遍存在的非生物胁迫, 它进化了各种性状, 其中一些已在生化或生理水平8,9,10, 11个。
自2010年以来, 有400多种同行更新的出版物将s. parvula作为目标物种, 或将其用于与其他植物基因组的比较。然而, 通过更仔细地研究已经进行了哪些类型的研究, 可以确定一个明显的瓶颈。这些报告大多讨论了在未来研究中的潜在用途, 或将其用于比较基因组或系统学研究。由于没有为s. parvula制定概念验证转换协议, 尽管迄今为止已将最高质量的植物基因组之一组装和组装, 但该协议尚未用于功能基因组研究。注释成染色体级的假分子1。
农杆菌介导的花浸转化方法已成为在塔利亚纳中最广泛使用的产生植物基因的方法, 开发可复制的转化系统是其成功的关键因素。遗传模型12,13。然而, 并非所有的布拉西卡科物种都已被证明是成功地转化使用了为a.thaliana 开发的花浸法。特别是包括白斑的 Brassicaceae 系 ii 种一直对以植物浸渍为基础的转化方法14,15。
由于罗非鱼开花习性不确定, 加上叶片形态狭窄, 采用标准农杆菌介导的花浸转化方法具有挑战性。在这项研究中, 我们报告了我们为s. parvula的可重复转化而开发的修改后的协议。
Protocol
1. 植物生长 种子灭菌 (可选) 在双蒸馏水 (ddh2o)中制备50% 的漂白剂, 在50毫升管中制备1或2滴非离子洗涤剂 (见材料表)。将管反转多次, 混合溶液。请注意:最好在具有紫外线灭菌表面的层流柜中进行种子灭菌15分钟。 在 1.5 ml 管中加入漂白剂溶液, 加入 ~ 100–200 s. parvula 种子。充分混合, 让管坐5分钟。 从试管中?…
Representative Results
我们开发了一个转化协议, 可以在150天内收获 t 0 种子, 使用的是从 a. thaliana的花浸法修改的方法.图 1显示了时间线和s. parvula 植物的摘要, 它们代表了通过花浸执行转换的最佳阶段。我们选择了在萌发后60-80天内具有70-80 朵花的植物作为转化的目标阶段。在这一阶段, 少量先前存在的开放或受精卵和花在植物浸润之前被花<em…
Discussion
植物的生理状态对转化效率有显著影响.利用健康而有活力的植物进行转化是成功转化的关键要求。与理想转化的健康植物相比, 水或光有压力的植物的花将更少 (图 1, 中心面板)。在低于 130μmol m-2 s-1 的光强下生长, 但植物往往更脆弱;这样的植物会导致更多的流产花后, 花浸水。帕鲁拉倾向于以高于塔利亚纳的速度…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家科学基金会 mcb 1616827 奖的支持。
Materials
| Agar | VWR International, Radnor, PA | 90000-762 | Bacto Agar Soldifying Agent, BD Diagnostics |
| B5 vitamins | Sigma-Aldrich, St. Louis, MO | G1019 | Gamborg’s Vitamin Solution |
| Desiccant | W A Hammond Drierite, Xenia, OH | 22005 | Indicating DRIERITE 6 mesh |
| Destination vector for plant transformation | TAIR | Vector:6531113857 | pKGWFS7 |
| Electroporation cuvette | USA Scientific | 9104-5050 | Electroporation cuvette, round cap, 0.2 cm gap |
| Electroporator | BIO-RAD Laboratories, Hercules, CA | 1652100 | MicroPulser Electroporator |
| Fertilizer beads | Osmocote Garden, Marysville, OH | N/A | Osmocote Smart-Release Plant Food Flower & Vegetable |
| Gel extraction kit | iNtRON Biotechnology, Boston, MA | 17289 | MEGAquick-spin Total fragment DNA purification kit |
| Gentamicin | Sigma-Aldrich, St. Louis, MO | G1914-5G | Gentamicin sulfate |
| Glufosinate-ammonium (11.3%) herbicide (BASTA) | Bayer environmental science, Montvale, NJ | N/A | FINALE herbicide |
| Kanamycin | VWR International, Radnor, PA | 200004-444 | Kanamycin monosulfate |
| MES | Bioworld, Dublin, OH | 41320024-2 | MES, Free Acid |
| MS salt | MP Biomedicals, Santa Anna, CA | 092621822 | Hoagland's modified basal salt mixture |
| N6-benzylaminopurine (BA) | Sigma-Aldrich, St. Louis, MO | B3274 | 6-Benzylaminopurine solution |
| NaCl | Sigma-Alrich | S7653 | Sodium chloride |
| Non-ionic detergent | Sigma-Aldrich, St. Louis, MO | 9005-64-5 | TWEEN 20 |
| Plasmid isolation kit | Zymo Research, Irvine, CA | D4036 | Zyppy Plasmid Kits |
| Recombinase enzyme mix kit | Life Technology | 11791-020 | Gateway LR Clonase II Enzyme mix |
| Rifampicin | Sigma-Aldrich, St. Louis, MO | R3501-1G | Rifampicin, powder, >= 97% (HPLC) |
| Shaking incubator | ThermoFisher Scientific, Waltham, MA | SHKE4450 | MaxQ 4450 Benchtop Orbital Shakers |
| Soil mix | Sun Gro | SUN239223328CFLP | Sun Gro Metro-Mix 360 Grower Mix |
| Spectinomycin | VWR International, Radnor, PA | IC15206705 | |
| Sterile 50ml conical tubes | USA Scientific, Ocala, FL | 1500-1811 | 50 ml conical screw cap tubes, copolymer, racks, sterile |
| Sucrose | VWR International, Radnor, PA | 57-50-1 | Sucrose, ACS |
| Surfactant solution | Lehle seeds, Round Rock, TX | VIS-02 | Silwet L-77 |
| Topoisomerase-based cloning kit | Life Technologies, Carlsbad, CA | K240020 | pENTR/D-TOPO Cloning Kit, with One Shot TOP10 Chemically Competent E. coli |
| Tryptone | VWR International, Radnor, PA | 90000-282 | BD Bacto Tryptone, BD Biosciences |
| Yeast Extract | VWR International, Radnor, PA | 90000-722 | BD Bacto Yeast Extract, BD Biosciences |
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Cite This Article
Wang, G., Pantha, P., Tran, K., Oh, D., Dassanayake, M. Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula. J. Vis. Exp. (143), e58544, doi:10.3791/58544 (2019).