Alonso Jose M, Stepanova Anna N
Department of Genetics, North Caroline State University, Raleigh, NC, USA.
Methods Mol Biol. 2014;1062:271-83. doi: 10.1007/978-1-62703-580-4_15.
The study of a gene's function requires, in many cases, the ability to reintroduce the gene of interest or its modified version back into the organism of choice. One potential caveat of this approach is that not only the coding region but also the regulatory sequences of a gene should be included in the corresponding transgenic construct. Even in species with well-annotated genomes, such as Arabidopsis, it is nearly impossible to predict which sequences are responsible for the proper expression of a gene. One way to circumvent this problem is to utilize a large fragment of genomic DNA that contains the coding region of the gene of interest and at least 5-10 kb of flanking genomic sequences. To facilitate these types of experiments, libraries harboring large genomic DNA fragments in binary vectors have been constructed for Arabidopsis and several other plant species. Working with these large clones, however, requires some special precautions. In this chapter, we describe the experimental procedures and extra cautionary measures involved in the identification of the clone containing the gene of interest, its transfer from E. coli to Agrobacterium, and the generation, verification, and analysis of the corresponding transgenic plants.
在许多情况下，对基因功能的研究需要有能力将感兴趣的基因或其修饰版本重新导入到所选的生物体中。这种方法的一个潜在问题是，相应的转基因构建体不仅应包含基因的编码区，还应包含其调控序列。即使在基因组注释良好的物种中，如拟南芥，几乎也不可能预测哪些序列负责基因的正确表达。解决这个问题的一种方法是利用大片段的基因组DNA，其包含感兴趣基因的编码区以及至少5-10kb的侧翼基因组序列。为便于进行这类实验，已经为拟南芥和其他几种植物构建了在二元载体中携带大片段基因组DNA的文库。然而，使用这些大克隆需要一些特殊的预防措施。在本章中，我们描述了在鉴定含有感兴趣基因的克隆、将其从大肠杆菌转移到农杆菌以及产生、验证和分析相应转基因植物过程中所涉及的实验程序和额外的注意事项。
