真菌快速缺失生产<em>农杆菌</em>介入OSCAR删除构造的转换
Summary
通过同源重组产生的基因缺失突变体是基因功能研究的黄金标准。描述了用于快速产生缺失构建体的OSCAR（农杆菌 – 重组 – 质粒的一步构建）方法。 土壤杆菌介导的真菌转化如下。最后，提出了基于真菌转化体基因缺失的基于PCR的确认方法。
Abstract
精确删除感兴趣的基因，同时使基因组的其余部分保持不变，为确定特定基因在活体中的功能提供了理想的产物。在本协议中，描述了精确快速删除质粒构建的OSCAR方法。 OSCAR依赖于进行单次重组酶反应的克隆系统，其中包含纯化的PCR扩增的目的基因的5'和3'侧翼和两个质粒pA-Hyg OSCAR（标记载体）和pOSCAR（组装体向量）。正确组装的缺失载体的确认通过限制性消化作图进行测序。然后使用根癌土壤杆菌介导将缺失构建体引入真菌孢子（简称ATMT）。最后，描述PCR测定以确定是否通过同源或非同源重组整合的缺失构建体，表明基因缺失或异位整合。这种方法已被成功地用于删除大丽花轮枝 菌属和其他物种中的尖角 镰孢属（Fusarium verticillioides）中的许多基因。
Introduction
遗传解剖是确定个体或基因组合的功能重要性的有力方法。了解特定基因作用的标准方法是在任何其他基因中不改变单基因突变体的产生。最强大和最不可能的混杂方法是完全和精确地删除感兴趣的基因开放阅读框架（GOI ORF）而不损害任何其他基因功能。
因为用于缺失质粒生成的标准连接方法需要多个步骤，OSCAR 1的合理性就是产生更快速的体外方法。 图1描述了OSCAR方法中的组装过程。本文描述的方法具有将单个多部分反应中的单个基因缺失载体的快速构建与随后的根癌土壤杆菌介导转录（ATMT）。 OSCAR非常快速，并且与其他策略（例如在酵母2中使用吉布森装配）相比较。 OSCAR方法成功应用于几种真菌菌根霉菌。这些物种包括： 镰孢镰孢 （未发表）， 大丽花 ， e 3 ，，ica ica ica ica ii ii ii ii ii ii um um um um um。。。。。 SP。 Vasinfectum 6 ，Pestalotiopsis microspora 7 ，Colletotrichum higginsianum 8 和Dothistroma septosporum 9和Sarocladium zeae （未发表） 。
该方案提供了包括引物设计，侧翼PCR扩增，OSCAR BP反应，缺失构建结构确认，转化的方法的逐步说明土壤杆菌的离子与构建物接着基于ATMT将缺失构建体转移到真菌细胞中，最后将真菌缺失突变体与具有异位整合的缺失构建体的突变体区分开。
Protocol
Representative Results
Discussion
已经成功地应用了农杆菌的一步构建 – 组合就绪质粒（OSCAR），其中越来越多的子囊菌真菌。假设农杆菌介导的转化和同源重组是可能的，该方法还应该容易地应用于担子菌门和其他真菌门（具有适当的启动子驱动选择性标记基因）的物种。已经产生了额外的标记载体以使抗真菌化合物的选择多样化，并且允许产生双重和高级突变体。这些包括抗G418，奈瑟菌素，最近除草剂草铵膦和?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢以下本科生和高中生在镰刀菌突变体中产生OSCAR突变体：Anjellica Miller，Athar Naseer，Xiu Lin，Katelyn Woodburry，Chelsea Patterson，Kathleen Robertson，Krystina Bradley，Ashton Rogers，Alexis McKensie，Manny Hernandez ，Ashli Crepsac，Jeff Delong，Christian King，Je Jeong，Maria Belding，Christy Burre，Daniel O'Meara，Lauren（Victoria）Cook，Jake Goodman，Sampriti De，Oge Okoye，Alyssa Beckstead，Garrett Hibbs，Nick Goldstein，Caroline Twum ，Chris Benson，Louis Stokes，Hannah Itell，Jane Hulse，Jasim Mohammed，James Loggins，Kelli Russell，Gre'Nisha Jones，Kristin Sheaffer，Mariam Hammady，Ava Wilson，Katrina Bazemore，Toney Harper，Karlin McGhee，Mohmed Momin，Rima Momin ，Thi Ngoc Le和Angel Pham。
Materials
| FungiDB | Database/ http://fungidb.org/fungidb/ | ||
| IDT PrimerQuest | IDT | Primer design online software/ http://www.idtdna.com/Primerquest/Home/Index | |
| Microsoft Word | Sequence file manipulation | ||
| Low Na LB Spec 100 medium | E. coli transformant selection, composition: 1% tryptone, 0.05% NaCl, 0.5% yeast extract, 1.5 % agar if for solid medium | ||
| Co-cultivation medium | ATMT transformation induction (Reference 12) | ||
| Aspergillus minimal medium with Hygromycin | Fungal transformant selection | ||
| PDA medium | Acumedia | 7149A | Single spore slant tubes |
| PDA-Hyg-Kan medium | Fungal ransformant isolation, PDA containing 150 μg/ml hygromycin B and 100 μg/ml Kanamycin; | ||
| Glass beads | Genlantis | C400100 | Plate spreading |
| Nitrocellulose filters (47mm) | Fisher | 09-719-555 | Co-culturing for ATMT |
| Various centifuge tubes | multiple preps | ||
| Petri plates (various) | Culturing of bacteria and Fungi | ||
| pA-Hyg OSCAR | Addgene | 29640 | Selectable marker vector |
| pOSCAR | Addgene | 29639 | Assembly vector |
| DH5a One Shot Competent E. coli cells | Life Technologies | 12297-016 | BP reaction transformation |
| ccdB survival E. coli cells | Life Technologies | A10460 | Maintenance of pOSCAR |
| Wooden transfer sticks | Colony streaking | ||
| Toothpicks | Colony picking | ||
| Microcentrifuge | Pelleting Bacteria etc | ||
| Preparative centrifuge | Fungal spore collection | ||
| Dissecting microscope | Single spore isolation | ||
| Automated Cell Counter | Spore suspension calculation | ||
| Compound microscope | Hemocytometer cell counting | ||
| QIAquick PCR Purification Kit | Qiagen | 28104 | PCR gene flank produict purification |
| TaKaRa LA Taq | Takara Bio USA | RR002A | Hi Fidelity taq polymerase for OSCAR flank generation |
| Hygromycin B | InvivoGen | ant-hg-5 | |
| Spectinomycin | Sigma | 22189-32-8 | |
| Cefotaxim | TCI America | C2224 | |
| Moxalactam | Sigma-Aldrich | 43963 | |
| GelRed | Phenix Research Products | RGB-4103 | Post staining agarose gels |
| Qiagen QIAquick PCR Purification Kit (Cat. No. 28104) | |||
| (OneShot_ Mach1TM T1R or One Shot_ OmniMAX™ 2 T1R from Invitrogen) | Thermo Fisher Scientific | C862003 | |
| Gateway BP Clonase II Enzyme mix | Thermo Fisher Scientific | 11789020 | Used to assemble deletion construct in pOSAR |
| PrimerQuest tool | IDT | Used in step 1.4; available on http://www.idtdna.com/Primerquest/Home/Index |
References
- Paz, Z., García-Pedrajas, M. D., Andrews, D. L., Klosterman, S. J., Baeza-Montañez, L., Gold, S. E. One step construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an efficient and robust tool for ATMT based gene deletion construction in fungi. Fungal Genet Biol. 48 (7), 677-684 (2011).
- Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., Smith, H. O. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods. 6 (5), 343-345 (2009).
- Klosterman, S. J., et al. Comparative genomics yields insights into niche adaptation of plant vascular wilt pathogens. PLoS Pathog. 7 (7), (2011).
- Xue, C., Wu, D., Condon, B. J., Bi, Q., Wang, W., Turgeon, B. G. Efficient gene knockout in the maize pathogen Setosphaeria turcica using Agrobacterium tumefaciens-mediated transformation. Phytopathology. 103 (6), 641-647 (2013).
- Xu, C., et al. A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii. PloS One. 9 (9), (2014).
- Crutcher, F. K., Liu, J., Puckhaber, L. S., Stipanovic, R. D., Bell, A. A., Nichols, R. L. FUBT, a putative MFS transporter, promotes secretion of fusaric acid in the cotton pathogen Fusarium oxysporum f. sp. vasinfectum. Microbiology. 161, 875-883 (2015).
- Yu, X., Wang, Y., Pan, J., Wei, D., Zhu, X. High frequency of homologous gene disruption by single-stranded DNA in the taxol-producing fungus Pestalotiopsis microspora. Ann Microbiol. 65 (4), 2151-2160 (2015).
- Korn, M., Schmidpeter, J., Dahl, M., Müller, S., Voll, L. M., Koch, C. A Genetic Screen for Pathogenicity Genes in the Hemibiotrophic Fungus Colletotrichum higginsianum Identifies the Plasma Membrane Proton Pump Pma2 Required for Host Penetration. PloS One. 10 (5), e0125960 (2015).
- Chettri, P. . Regulation of dothistromin toxin biosynthesis by the pine needle pathogen Dothistroma septosporum: a thesis presented in the partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatu, New Zealand . , (2014).
- Chen Zhou, ., Yujun Yang, ., Jong, A. Y. Mini-prep in ten minutes. Biotechniques. 8 (2), 172 (1990).
- Sanger, F., Nicklen, S., Coulson, A. R. DNA sequencing with chain-terminating inhibitors. P Natl Acad SciUSA. 74 (12), 5463-5467 (1977).
- Khang, C. H., Park, S. Y., Rho, H. S., Lee, Y. H., Kang, S., Wang, K. a. n. Filamentous fungi (Magnaporthe grisea and Fusarium oxysporum). Agrobacterium Protocols. 2, 403-420 (2007).
- Zhang, Y. J., Zhang, S., Liu, X. Z., Wang Wen, H. A., M, A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains. Lett Appl Microbiol. 51 (1), 114-118 (2010).
- Pluthero, F. G. Rapid purification of high-activity Taq DNA polymerase. Nucleic Acids Res. 21 (20), 4850-4851 (1993).
- McCluskey, K. Boosting Research and Industry by Providing Extensive Resources for Fungal Research. Gene Expression Systems in Fungi: Advancements and Applications. , 361-384 (2016).
