通过农杆菌介导的BiBAC修饰及向玉米(Zea mays) Hi-II未成熟胚的稳定转移

Q1 Agricultural and Biological Sciences Current protocols in plant biology Pub Date : 2018-02-13 DOI:10.1002/cppb.20061

Jon P. Cody, Nathaniel D. Graham, James A. Birchler

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引用次数: 5

摘要

二元细菌人工染色体(BiBAC)是大型插入克隆载体，包含农杆菌介导转化所需的必要特征。然而，在大肠杆菌(DH10B)和农杆菌(Agrobacterium tumefaciens)中BiBACs的大尺寸和低拷贝数使得克隆实验比其他可用的二元载体系统更加困难。因此，一个概述高分子量(HMW)构建物的制备、修饰和转化的方案对于希望在植物基因组学研究中使用bibac的研究人员是有利的。本单元不包括将HMW DNA克隆为BiBAC载体。希望将HMW DNA克隆到BiBACs中的研究人员可以参考Zhang等人(2012;doi: 10.1038 / nprot.2011.456)。©2017 by John Wiley &儿子,Inc。

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BiBAC Modification and Stable Transfer into Maize (Zea mays) Hi-II Immature Embryos via Agrobacterium-Mediated Transformation

Binary Bacterial Artificial Chromosomes (BiBAC) are large insert cloning vectors that contain the necessary features required for Agrobacterium-mediated transformation. However, the large size of BiBACs and low-copy number in Escherichia coli (DH10B) and Agrobacterium tumefaciens make cloning experiments more difficult than other available binary vector systems. Therefore, a protocol that outlines preparation, modification, and transformation of high-molecular weight (HMW) constructs is advantageous for researchers looking to use BiBACs in plant genomics research. This unit does not cover the cloning of HMW DNA into BiBAC vectors. Researchers looking to clone HMW DNA into BiBACs can refer to Zhang et al. (2012; doi: 10.1038/nprot.2011.456). © 2017 by John Wiley & Sons, Inc.

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Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.