摘要
经转座子Tn5诱变,获得20株紫云英根瘤菌菌株109胞外多糖(Exopolysaccharide,EPS)缺陷型(EPS-deficient,Exo^-)变种。这些变种仍都是原养型,在含不同的碳底物或不同浓度碳底物的固体培养基上,菌落型态均无改变。与亲本菌相比,变种的EPS产量明显降低。所有变种未能在其宿主植物紫云英上结瘤。利用载体质粒pMN2,经Exo^-变种与大肠杆菌受体菌交配,通过选择Tn5卡那霉素抗性标记的转移,构建成带有Tn5及菌株109多糖基因DNA片段的R-prime质粒。R-prime质粒能稳定地存在于菌株109中。大部分变种的Exo^-表型能被R-prime质粒互补,但R-prime质粒未能使这些Exo^-变种恢复有效结瘤的共生能力。根据互补结果,20株Exo^-变种分为6种不同的互补群,其中5种互补群的多糖位点在遗传上是连锁的。
Twenty exopolysaccharide (EPS)-deficient (Exo^-) mutants wereisolated after Rhizobium astragalus strain 109 was subjected to transposonTn5 mutagenesis. These mutants had similar colony morphologies andtheir phenotype was not altered by growing on the media supplementedwith different carbohydrates or different concentrations of carbohydrate.The EPS yields of the mutants were dramatically decreased which wereabout 0.5% to 36% of wild-type levels of EPS. None of the EXo^- mu-tants were able to nodulate their host plant, Astragalus sinicus. R-prime plasmids carrying regions of strain 109 coding for exopoly-saccharide synthesis were isolated from plasmid PMN_2,a kanamycinsen-sitive derivative of plasmid R68 .45. The R-primes were obtained from matings between Tn5-induced Exo^- mutants of strain 109 and E.coli.recipients by selecting for the kanamycin resistance marker of Tn5. Itwas shown that the regions inserted into the R-primes were identicalwith those DNA segments where Tn5 was located in the respectiveparental Exo^- mutant. The R-primes were stable in strain 109 back-ground. R-primes were able to complement the Exo^- phenotype of mostmutants. Complementation of these Exo^- mutants. did not restoredtheir symbiotic abilities of effective nodulation. Strain 109 containingplasmid pMN2 or Tn5 was also unable to nodulate A. sinicus. The 20Exo^- mutants were classified into six distinct complementation groupsbased on complementation data. Five of the six exo loci were geneti-cally linked.
出处
《生物工程学报》
CAS
CSCD
北大核心
1991年第2期124-131,共8页
Chinese Journal of Biotechnology
关键词
根瘤菌
胞外多糖合成
遗传分析
Rhizobium
transposon mutagenesis
exopolysaccharide synthesis
genetic analysis
