用 BIBAC 二元矢量生成单拷贝插入的转基因植物
Summary
使用 pBIBAC 的二进制向量, 可以生成具有完整单拷贝插入的转基因植物, 这是一个简单的过程。在这里, 提出了一系列的协议, 指导读者通过生成转基因拟南芥植物的过程, 并测试植物的完整性和复制数量的插入。
Abstract
在生成转基因植物时, 一般的目的是要有稳定的转基因表达。这需要一个单一的, 完整的整合的转基因, 因为多拷贝集成往往受到基因沉默。与其他 pBIBAC 衍生物一样, 基于细菌人工染色体 (pBIBAC) 的网关兼容二进制向量允许单拷贝转基因的高效插入。作为对原 pBIBAC 的改进, 一个网关盒已被克隆成 pBIBAC, 因此, 现在的兴趣序列可以很容易地纳入矢量转移 dna (T DNA) 的网关克隆。通常情况下, 与 pBIBAC 的转换结果是0.2–0.5% 的效率, 即转基因的一半携带完整的单拷贝集成的 T DNA。pBIBAC 的载体是可利用的抗膦铵或 DsRed 荧光的种子大衣, 以选择在植物, 和抗卡那霉素作为选择的细菌。在这里, 提出了一系列的协议, 引导读者通过使用 pBIBAC 生成转基因植物的过程: 从将兴趣序列重新结合到 pBIBAC 的选择向量, 到植物转化与农杆菌, 选择转基因, 并测试植物的完整性和复制数量的插入使用 DNA 印迹。注意设计一种 DNA 印迹策略, 以识别单个和多个位点的单拷贝和多复制集成。
Introduction
在生成转基因植物时, 通常的目的是使综合转基因 (s) 稳定表达。这可以通过转基因的完整的单拷贝集成来实现。多重整合可以导致基因表达的增加, 同时也会使转基因沉默。如果插入的序列排列在串联或反转重复的1、2、3、4中,则更有可能转基因沉默。在农杆菌介导的转化实验中, 二进制向量被用作航天飞机, 将感兴趣的序列传递到植物基因组中。集成到植物基因组的数量依赖于农杆菌5、6中二进制向量的拷贝号.许多常用的二进制向量都是高拷贝向量, 因此产生高平均的转基因拷贝数:拟南芥5中的3.3 到4.9 个拷贝。
通过使用.BIBAC 中具有低拷贝数的二进制向量 (如7) 或从a. 农杆菌染色体5启动 t dna, 可以降低 t dna 积分的数量。在这种情况下, 转基因集成的平均数量低于 25,8,9,10。由于在A. 农杆菌和大肠杆菌中都是单拷贝, BIBAC 衍生物可以维护和交付 150 kb11大的构造。
与 GW 兼容的 BIBAC 矢量10,12允许使用网关克隆轻松地将感兴趣的基因引入到向量中。网关技术的使用大大简化了克隆过程, 也克服了与大型低拷贝数向量13、14相关的常见问题, 如低 DNA 产量和有限的独特限制选择。可用于克隆7、11的站点。在植物 (图 1)10,12中, pBIBAC 的衍生物可在种子大衣 (pBIBAC-RFP) 中对膦铵 (pBIBAC 棒) 或 DsRed 荧光进行抗性。对于这两种载体, 卡那霉素抗性基因被用作细菌的选择标记。
pBIBAC 的向量结合: (1) 易于设计和遗传操作在大肠杆菌和 (2) 完整的单拷贝集成在 planta高效率。在拟南芥中, pBIBAC 的向量平均1.7 积分, 其中大约一半的转基因植株携带着单个集成的 T 型 DNA10。
转基因的稳定表达是大多数转基因产生的必要条件。稳定的转基因表达可以通过完整的单拷贝集成实现。然而, 与转基因植物进行完整的, 单拷贝集成是更重要的, 如果例如, 目的是研究的效率, 染色质的过程, 如诱变, 重组, 或修复, 并依赖这些在插入部位的基因组位置和染色质结构的过程。为了我们的兴趣, 研究寡核苷酸定向诱变 (ODM) 对局部基因组上下文的依赖性, 生成了一组完整的、单拷贝的突变报告基因集成的报告 (图 2)10。使用这组线, 结果表明, 在不同基因组位置集成的转基因基因座的 ODM 效率不同, 尽管转基因表达水平相当相似。
Protocol
1. 将感兴趣的序列插入二进制向量中 准备网关条目和二进制向量。 根据供应商的建议, 将包含 DNA 片段或感兴趣基因的网关入口向量隔离。注: BIBAC-GW 载体需要使用卡那霉素 (公里) 来选择细菌, 因此, 使用一个输入向量与另一种抵抗标记而不是卡那霉素。例如, 携带庆大霉素抗性基因的 pENTR 转基因载体是一个很好的选择12。 传播和隔离 BIBAC …
Representative Results
利用 BIBAC 系统, 生成了用于研究植物 ODM 的记者构造10。构造是在网关条目向量 pENTR-gm12中设计的, 并使用网关 LR 复合反应插入 pBIBAC BAR (图 1)。 拟南芥是与 pDM19, 一个 BIBAC-GW 质粒与 mTurquoise eYFP 的记者, 携带一个平移停止密码子在 eYFP 阅读框架的位置 120 (mTurquoise2-eYFP*40) …
Discussion
对产生转基因的关键是单一的, 完整的整合的转基因是使用二进制向量的选择。BIBAC 家族载体已被用来交付许多植物物种的利益序列23,24,25,26,27,28。BIBAC 向量, 包括 BIBAC, 具有高效率的单拷贝集成: 每行插入的平均数量是1.5 到 2, 而最常用的二进制向量…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项研究得到荷兰技术基金会 STW (12385) 的支持, 该基金是荷兰科学研究组织 (NWO) 的一部分, 由经济事务部 (检察官办公室赠款12385至 MS) 部分资助。 我们感谢卡罗尔 m. 汉密尔顿 (美国康奈尔大学) 提供 pCH20, BIBAC 的主干。
Materials
| Kanamycin sulphate monohydrate | Duchefa | K0126 | |
| Gentamycin sulphate | Duchefa | G0124 | |
| Rifampicin | Duchefa | R0146 | |
| Tetracycline hydrochloride | Sigma | T-3383 | |
| DB3.1 competent cells | Thermo Scientific – Invitrogen | 11782-018 | One Shot ccdB Survival 2 T1R Competent Cells (A10460) by Invitrogen or any other ccdB resistant E. coli strain can be used instead |
| DH10B competent cells | Thermo Scientific – Invitrogen | 18290-015 | |
| Gateway LR clonase enzyme mix | Thermo Scientific – Invitrogen | 11791-019 | |
| tri-Sodium citrate dihydrate | Merck | 106432 | |
| Trizma base | Sigma-Aldrich | T1503 | |
| EDTA disodium dihydrate | Duchefa | E0511 | |
| Proteinase K | Thermo Scientific | EO0491 | |
| Bacto tryptone | BD | 211705 | |
| Yeast extract | BD | 212750 | |
| Sodium chloride | Honeywell Fluka | 13423 | |
| Potassium chloride | Merck | 104936 | |
| D(+)-Glucose monohydrate | Merck | 108346 | |
| Electroporation Cuvettes, 0.1 cm gap | Biorad | 1652089 | |
| Electroporator Gene Pulser | BioRad | ||
| Magnesium sulfate heptahydrate | Calbiochem | 442613 | |
| D(+)-Maltose monohydrate 90% | Acros Organics | 32991 | |
| Sucrose | Sigma-Aldrich | 84100 | |
| Silwet L-77 | Fisher Scientific | NC0138454 | |
| Murashige Skoog medium | Duchefa | M0221 | |
| Agar | BD | 214010 | |
| Glufosinate-ammonium (Basta) | Bayer | 79391781 | |
| Restriction enzymes | NEB | ||
| Ethidium Bromide | Bio-Rad | 1610433 | |
| Electrophoresis system | Bio-Rad | ||
| Sodium hydroxide | Merck | 106498 | |
| Hydrochloric acid | Merck | 100316 | |
| Blotting nylon membrane Hybond N+ | Sigma Aldrich | 15358 | or GE Healthcare Life Sciences (RPN203B) |
| Whatman 3MM Chr blotting paper | GE Healthcare Life Sciences | 3030-931 | |
| dNTP | Thermo Fisher | R0181 | |
| Acetylated BSA | Sigma-Aldrich | B2518 | |
| HEPES | Sigma-Aldrich | H4034 | |
| 2-Mercaptoethanol | Merck | 805740 | |
| Sephadex G-50 Coarse | GE Healthcare Life Sciences | 17004401 | or Sephadex G-50 Medium (17004301) |
| Dextran sulfate sodium salt | Sigma-Aldrich | D8906 | |
| Sodium Dodecyl Sulfate | US Biological | S5010 | |
| Salmon Sperm DNA | Sigma-Aldrich | D7656 | |
| Sodium dihydrogen phosphate monohydrate | Merck | 106346 | |
| Storage Phosphor screen and casette | GE Healthcare Life Sciences | 28-9564-74 | |
| Phosphor imager | GE Healthcare Life Sciences | Typhoon FLA 7000 | |
| UV Crosslinker | Stratagene | Stratalinker 1800 | |
| cling film (Saran wrap) | Omnilabo | 1090681 | |
| Agarose | Thermo Scientific – Invitrogen | 16500 | |
| Boric acid | Merck | 100165 | |
| DNA marker ‘Blauw’; DNA ladder. | MRC Holland | MCT8070 | |
| DNA marker ‘Rood’; DNA ladder | MRC Holland | MCT8080 | |
| Hexanucleotide Mix | Roche | 11277081001 | |
| Large-Construct Kit | Qiagen | 12462 | |
| Heat-sealable polyethylene tubing, clear | various providers | the width of the tubing should be wider than that of blotting membrane | |
| Heat sealer | |||
| Membrane filter disk | Merck | VSWP02500 | |
| Magnesium chloride | Merck | 105833 | |
| Hybridization mesh | GE Healthcare Life Sciences | RPN2519 |
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Citar este artículo
Tark-Dame, M., Weber, B., de Sain, M., Anggoro, D. T., Bader, R., Walmsley, A., Oka, R., Stam, M. Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector. J. Vis. Exp. (133), e57295, doi:10.3791/57295 (2018).