Zhou B, Huang J, Su W, Song H
Shanghai Institute of Plant Physiology, Chinese Academy of Sciences, China.
Chin J Biotechnol. 1998;14(1):37-43.
pJB-B5 was a recombinant plasmid of pRK415 containing the 5.9 kb B5 fragment from exoR'-11. After pJB-B5 was mutagenized by MT614 (mal::Tn5), 10 plasmids TN1-1, TN1-12, TN2-2, TN2-3, TN3-1, TN4-1, TN9-1, TN10-1, TN13-1, and TN14-1 with different Tn5 insertions in the B5 fragment were constructed. By conjugation of TN1-1, etc., into Rhizobium huakuii 107 containing the P-group plasmid pPH1JI which is incompatible with pRK415, and simultaneous selection for Rifr (conferred by strain 107), Gm(r) (conferred by pPH1JI), and Nmr (retention of Tn5), R. huakuii 107 transconjugant yields a strain in which Tn5 has recombined into the R. huakuii 107 genome. Three EPS-deficient (Exo-) mutants 107 (TN2-2), 107 (TN10-1), and 107 (TN13-1) were isolated and their inserted Tn5 was certified as the result of a double homologous recombinant event by Southern hybridization analysis. This result showed the Tn5 region-directed mutagenesis is an efficient way to select Exo- mutant in R. huakuii.
pJB - B5是一种pRK415重组质粒，含有来自exoR'-11的5.9 kb B5片段。pJB - B5经MT614（mal::Tn5）诱变后，构建了10个在B5片段中有不同Tn5插入的质粒TN1 - 1、TN1 - 12、TN2 - 2、TN2 - 3、TN3 - 1、TN4 - 1、TN9 - 1、TN10 - 1、TN13 - 1和TN14 - 1。通过将TN1 - 1等与含有与pRK415不相容的P组质粒pPH1JI的华癸根瘤菌107进行接合，并同时选择Rifr（由菌株107赋予）、Gm(r)（由pPH1JI赋予）和Nmr（Tn5的保留），华癸根瘤菌107转接合子产生了一个Tn5已重组到华癸根瘤菌107基因组中的菌株。分离出三个EPS缺陷（Exo -）突变体107（TN2 - 2）、107（TN10 - 1）和107（TN13 - 1），通过Southern杂交分析证实其插入的Tn5是双同源重组事件的结果。该结果表明，Tn5区域定向诱变是在华癸根瘤菌中筛选Exo -突变体的有效方法。
