Simoens C, Alliotte T, Mendel R, Müller A, Schiemann J, Van Lijsebettens M, Schell J, Van Montagu M, Inzé D
Nucleic Acids Res. 1986 Oct 24;14(20):8073-90. doi: 10.1093/nar/14.20.8073.
The transformation of mutant plants with a complete recombinant library derived from wild-type DNA followed by assay of transformed plants for complementation of the mutant phenotype is a promising method for the isolation of plant genes. The small genome of Arabidopsis thaliana is a good candidate for attempting this so-called shotgun transformation. We present the properties of an A. thaliana genomic library cloned in a binary vector, pC22. This vector, designed to introduce genomic libraries into plants, contains the oriV of the Ri plasmid pRiHR1 by which it replicates perfectly stably in Agrobacterium. Upon transfer of the library from E. coli to A. tumefaciens large differences in transfer efficiencies of individual recombinant clones were observed. There is a direct relation between transfer efficiency and stability of the recombinant clones both in E. coli and A. tumefaciens. The stability is independent of the insert size, but seems to be related to the nature of the insert DNA. The feasibility of shotgun transformation and problems of statistical sampling are discussed.
用源自野生型DNA的完整重组文库转化突变植物，然后检测转化植物的突变表型互补情况，是分离植物基因的一种很有前景的方法。拟南芥的小基因组是尝试这种所谓鸟枪法转化的良好候选对象。我们展示了克隆于二元载体pC22中的拟南芥基因组文库的特性。该载体设计用于将基因组文库导入植物，含有Ri质粒pRiHR1的oriV，借此它能在农杆菌中极其稳定地复制。当文库从大肠杆菌转移至根癌农杆菌时，观察到各个重组克隆的转移效率存在很大差异。在大肠杆菌和根癌农杆菌中，转移效率与重组克隆的稳定性之间存在直接关系。稳定性与插入片段大小无关，但似乎与插入DNA的性质有关。本文讨论了鸟枪法转化的可行性以及统计抽样问题。
