利用Tn5定位诱变筛选紫云英根瘤菌Exo^-变种

Selection of EPS defecient Mutants (Exo -) from Rhizobiun huakuii 107 by Tn5 Region directed Mutagenesis

利用Ｔｎ５定位诱变方法，对质粒ｐＪＢＢ５进行Ｔｎ５插入诱变，得到１０个Ｔｎ５在５９ｋｂＢ５外源片段上有不同插入位点的质粒ＴＮ１１，ＴＮ１１２，ＴＮ２２，ＴＮ２３，ＴＮ３１，ＴＮ４１，ＴＮ９１，ＴＮ１０１，ＴＮ１３１，ＴＮ１４１。将Ｔｎ１１等分别转移到已经含有不相容质粒ｐＰＨ１ＪＩ的紫云英根瘤菌１０７菌株中，使之发生同源变换。通过抗性选择及表型鉴别，筛选到３株菌落表型干燥（Ｍｕｃ－）的酸性胞外多糖（ＥＰＳ）合成缺陷菌株（Ｅｘｏ－）１０７（ＴＮ２２），１０７（ＴＮ１０１），１０７（ＴＮ１３１）。Ｓｏｕｔｈｅｒｎ杂交分析证明这３株变种的Ｔｎ５插入确实是同源交换而不是转座产生，表明经过适当改良的Ｔｎ５定位诱变法可以应用于紫云英根瘤菌Ｅｘｏ－变种的筛选。

Recombinant plasmid pJB B5 was mutagenized by MT614(mal∷Tn5) and 10 plasmids TN1 1,TN1 12,TN2 2,TN2 3,TN3 1,TN4 1,TN9 1,TN10 1,TN13 1,TN14 1 with different Tn5 insertion in the 5.9kb foreign fragment were constructed.By conjugation of TN1 1 etc.into Rhizobium huakuii 107 containing the P group plasmid pPH1JI which is incompatable with pRK415 and stimultaneous selection for Rf r (conferred by strain 107) Gm r (conferred by pPH1JI) Nm r (retention of Tn5), R.huakuii 107 transconjugant yields strain in which Tn5 has recombined into R.huakuii 107 genome.Three EPS defecient(Exo -) mutants 107(TN2 2),107(TN10 1),107(TN13 1) were isolated and their inserted Tn5 was certified as the result of a double homologous recombinant event by Southern hybridization anaysis.This result showed the Tn5 region directed mutagenesis is a efficient way to select for Exo - mutant in R.huakuii