Rapid and efficient selection of recombinant site-directed mutants of Bradyrhizobium japonicum by colony hybridization.
Rapid and efficient selection of recombinant site-directed mutants of Bradyrhizobium japonicum by colony hybridization.
复制标题
通过菌落杂交快速有效地选择日本慢生根瘤菌重组定点突变体。
DOI：
10.1111/j.1574-6968.1993.tb06139.x
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发表时间：
1993
影响因子：
2.1
通讯作者：
Maier,RJ
中科院分区：
文献类型：
--
作者：
Fu,C;Maier,RJ
由于慢生型大豆根瘤菌具有较高的自发耐药性和生长缓慢的特点，定点突变体的筛选十分繁琐和耗时。建立了一种快速筛选B. japonicum发达。卡那霉素（Km）和壮观霉素（Sp）盒各自用于通过同源重组替换染色体中的DNA片段。该方法的主要新特征包括用于抗生素（Km或Sp）抗性突变体的简单平板选择，然后菌落划线，以及用于在硝酸纤维素过滤器上进行DNA杂交的裂解，从而能够直接鉴定重组定点突变体。这种方法使我们能够快速，方便地确定大量的阳性重组突变体从大量indicidual菌落。该方法消除了首先从每个突变体分离基因组DNA用于Southern杂交的需要。证实来自该方法的所有测试的定点突变体均表现出预期的突变体表型。
Due to the high incidence of spontaneous antibiotic resistance and slow growth ofBradyrhizobium japonicumstrains, screening for site-directed mutants is cumbersome and time-consuming. A rapid method for selection of recombinant site-directed mutants ofB. japonicumwas developed. A kanamycin (Km) and a spectinomycin (Sp) cassette were each used to replace DNA fragments in the chromosome by homologous recombination. The primary new features of this method involve a simple plate selection for the antibiotic (Km or Sp) resistant mutants, then colony streaking, and lysis for DNA hybridization on a nitrocellulose filter enabling direct identification of the recombinant site-directed mutants. This method has permitted us to quickly and easily identify a large number of positive recombinant mutants from a large number of indicidual colonies. The procedure eliminates the need to first isolate genomic DNA from each mutant for Southern hybridization. All of the tested site-directed mutants from this method were confirmed to exhibit the expected mutant phenotype.