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The function of a lysogenic rhizobiophage in the symbiosis between Rhizobium and leguminous plants.

溶原性根瘤噬菌体在根瘤菌和豆科植物共生中的功能。

基本信息

批准号：
04804056
负责人：
UCHIUMI Toshiki
金额：
$ 1.09万
依托单位：
Kagoshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04804056/
关键词：
Rhizobium lysogenic phage chromosome integration nod-genes attP attB site specific recombination ベクター

项目摘要

The temperate phage PHIU lysogenizes Rhizobium leguminosarum bv.trifolii 4S by site specific recombination between phage (attP) and bacterial (attB) sites. The attP site was identified on a 6.0 kb EcoRI fragment of the phage DNA and cloned into the EcoRI site on the suicide plasmid pSUP202. Plasmid pCI6, the resulting recombinant plasmid, could integrate into the chromosome of strain 4S at high frequency of 4.40 X 10^<-3>. The pCI6 could also integrate into the chromosome of R.leguminosarum bv.viciae K5 which was not sensitive to phage PHIU infection. Southern hybridization probed with attP fragment revealed that the integration site of pCI6 on the chromosome was completely identical between strains 4S and K5. It is suggested that pCI6 may be a useful chromosome integrative vector for gene transfer on Rhizobium.The fusion plasmid carrying the attP of phage PHIU and the nod-genes derived from R.leguminosarum bv.trifolii was constructed based on pSUP203 and designated as pCINodl. E.coli S17-1 harboring pCINod1 was conjugated with strain H1, a symbiotic defective mutant of strain 4S.The inoculation of the mating mixture resulted in nodule formation on the roots of white clover (Trifolium repens cv.Ladino). The nodules were observed by transmission electron microscopy. Rhizobium invaded into the host plant cells through infection threads and changed their form to bacteroids. Transconjugants were isolated from the nodules and one of them was designated as strain CNH1. Southern hybridization probed with attP revealed that pCINod1 integrated into the chromosome of strain CNH1 as same as pCI6. These results indicate that nod-genes integrated into the chromosome functioned efficiently in Rhizobium.

温带噬菌体PHIU溶菌豆科根瘤菌。噬菌体（attP）与细菌（attB）位点特异性重组。在噬菌体DNA的一个6.0 kb EcoRI片段上鉴定了attP位点，并将其克隆到自杀质粒pSUP202的EcoRI位点上。重组质粒pCI6能以4.40 × 10^<-3>的高频率整合到菌株4S的染色体上。pCI6也可以整合到豆科植物的染色体中。对噬菌体PHIU感染不敏感的毒株K5。利用attP片段进行南杂交，发现菌株4S和菌株K5染色体上pCI6的整合位点完全相同。提示pCI6可能是一种有效的根瘤菌基因转移的染色体整合载体。噬菌体PHIU的attP与豆科致病菌nod基因的融合质粒。在pSUP203的基础上构建了trifolii，命名为pCINodl。携带pCINod1的大肠杆菌S17-1与菌株4S的共生缺陷突变株H1结合。接种配种混合物可在白三叶草（Trifolium repens cv.Ladino）根部形成根瘤。透射电镜观察结节。根瘤菌通过感染线侵入寄主植物细胞，将其形态转化为类细菌。从结核中分离到转缀合物，其中一个被命名为菌株CNH1。利用attP进行Southern杂交，发现pCINod1与pCI6一样可以整合到菌株CNH1的染色体上。这些结果表明，整合到染色体中的节点基因在根瘤菌中发挥了有效的作用。