@article{oai:niigata-u.repo.nii.ac.jp:00004076,
 author = {皆川, 律子 and 磯田, 香保理 and 大竹, 憲邦 and 山田, 真也 and 五十嵐, 太郎 and 南沢, 究 and 大山, 卓爾},
 issue = {2},
 journal = {日本土壌肥料學雜誌, 日本土壌肥料學雜誌},
 month = {Apr},
 note = {大腸菌由来のβーグルクロニダーゼ(GUS)遺伝子gusAを導入したダイズ根粒菌Bradyrhizobium japonicum 61A124a株は,基質であるX-Glucを与えると,単生・共生のいずれの状熊においても青色のGUS代謝産物を菌体内に集積した.このGUS代謝産物をフェノールで抽出して吸光度を測定することにより土壌中のgus導入菌数の推定を試みた.根粒菌は土壌粒子に強く接着し,分離困難であったため,液体フェノールによって菌体を破壊してGUS代謝産物を抽出し,フェノール層の波長645nmの吸光度を測定した.菌密度とフェノール抽出液の吸光度には直線関係が認められた.土壌中の菌数測定には,土壌ごとに検量線を作成することにより吸光度から菌密度の推定が可能であった.なお,根粒菌の増殖はX-Glucの溶媒として用いたN,N-ジメチルホルムアミドとGUS代謝産物によって抑えられることから, GUS反応中における根粒菌数はー定に保たれていた.プラスチックケースに各種土壌を詰めてダイズを栽培し,gus導入菌を局所接種して接種菌の分布と根粒形成を判定した.その結果,根粒菌の増殖速度や分布は土壌によって違いが見られた.土着菌が多く生息する土壌では,接種菌の増殖率が高いにもかかわらず,根粒感染率は低かうた.一方,士着根粒菌密度の低い土壌では,接種菌の増殖率が低くても大部分の根粒が接種菌により形成されていた., Bradyrhizobium japonicum strain 61 A 124 a, in which Escherichia coli gus A gene was introduced to Bradyrhizobium japonicum, absorbs and hydrolyzes GUS-substrate (X-Gluc), and precipitates an indigo pigment. We attempted to use the absorbance of the accumulated GUS-metabolite as an indicator of the rhizobial number in soil. The accumulated indigo blue metabolite in rhizobia were extracted by phenol-water after they are incubated with X-Gluc (20 μL of 1% X-Gluc solution in N,N-dimethylformamide was added to 1 mL of culture solution) for 4 d. The absorbance of the extracted blue pigment in the phenol layer was measured at 645 nm. The initial number of rhizobia and the absorbance of the GUS-metabolite was positively correlated either in cultured 61 A 124 a or in the various types of soil inoculated with 61 A 124 a strain. In the presence of soil, the absorbance per rhizobial number was low compared with the pure culture. Therefore, a standard curve between rhizobial number of 61 A 124 a and the absorbance should be made for every soil sample. During incubation with X-Gluc, rhizobia did not proliferate because of the depression effect on cell growth of N,N-dimethylformamide and the accumulation of indigo pigment. The proliferation and mobility of inoculated 61 A 124 a strain was examined in a rhizobox containing various soil types into which soybeans were planted. The distribution and movement of the marked strain depended on the soil type. In the absence or very low density of indigenous Bradyrhizobium japonicum, the nodule was exclusively formed by the strain 61 A 124 a. On the other hand, the major part of nodules were formed by indigenous strains in the soil types containing a high population of indigenous bradyrhizobia, although the inoculated strain proliferated very well.},
 pages = {148--155},
 title = {GUS代謝産物のフェノール抽出による土壌中のgus導入根粒菌数の推定},
 volume = {68},
 year = {1997}
}