紫云英根瘤菌107菌株exo基因簇非连锁突变位点的克隆

用鸟枪法从3株紫云英根瘤菌107菌株的胞外多糖合成缺陷变种（Exo-）NA－05、NA－07和NA－08中克隆获得含有107菌株exo基因及Tn5的exo::Tn5片段。以pRK415为载体构建107菌株EcoRI酶切后DNA片段的部分基因库，用exo::Tn5做探针原位杂交得到一个阳性克隆。该克隆的外源片段4．2kb能恢复3个变种的多糖表型及结瘤固氮能力。酶切分析和Southern杂交表明，3株变种中Tn5插入位点相近。

Cloning of an Unlinked exo Site from EPS Synthesis Deficient Mutants of Rhizobium astragali Strain 107

Three exopolysaccharide-deficient mutants NA-05, NA-07 and NA-08, belnging to the F complementing groupwere subjected to the shotgun cloning method. Ten eco:: Tn5 fragments of abut 10 kb were cloned in pUC18 (Fig. 1). Analysis showed that they were identical and all cnsisted f a 4. 2 kb R. asraghi 107 fragment plus the 5. 8 kb Tn5 insertin (Fig. 2). After constructing a partial genomic library of R. asraopi 107 using plasmid pRK415,one clne the pRKM578 was isolated by using the pRK8f, one of exo::Tn5 clones as a probe. The pRKM578,containing a 4. 2 kb R. twaghi 17 DNA fragment, was mapped. Plant tests showed that the transcnjugants of pRKM578 and NA-05, NA-07 and NA-08 restored the EPS phentype of these three mutunts, as well as their nodulation and nitrogen fixatin abilities.By complementation test f NA05, NA-07 and NA-08 with plasmids carrying eco genes of R. meliloti and Rhizobium sp. NGR234, the functin of the exo gene cloned in pRK415 (the pRKM578) was identified as encoding for UDP-glucose-4-epimerase, as the exoB of R. meliloti and eroC f R. sp.NGR234.