| Project/Area Number |
04804056
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
植物生理学
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| Research Institution | Kagoshima University |
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Principal Investigator |
UCHIUMI Toshiki Kagoshima University, Department of Biology, Research Asistant, 理学部, 助手 (20193881)
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| Co-Investigator(Kenkyū-buntansha) |
ABE Mikiko Kagoshima University, Department of Biology, Asistant Professor., 理学部, 助教授 (00107856)
HIGASHI Shiro Kagoshima University, Department of Biology, Professor., 理学部, 教授 (60041216)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1992: ¥700,000 (Direct Cost: ¥700,000)
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| Keywords | Rhizobium / lysogenic phage / chromosome / integration / nod-genes / attP / attB / site specific recombination / ベクター |
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| Research Abstract |
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.
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