초록 ▼

질소 고정세균의 유전연구를 위한 기본 연구로 pRD1의 conjugation 빈도를 조사한 결과 대장균은 5 * 10￣²,Klebsiella균은 5 * 10￣6 이었으나 Rhizovium균은 conjugation이 일어나지 않았다. 또한 염색체의 질소고정 변이주와 plasmid 질소고정 변이주의 유전적 특성을 조사하였다. 한편 gene fusion 및 nif 유전자 조절에 관한 연구로 ΦMud1 과 Mucts62를 이용하여 얻어진 nif-lac fused 변이주는 NH₄+ 전재하에서 현저한 β-galactosidase 활성도 감소를

질소 고정세균의 유전연구를 위한 기본 연구로 pRD1의 conjugation 빈도를 조사한 결과 대장균은 5 * 10￣²,Klebsiella균은 5 * 10￣6 이었으나 Rhizovium균은 conjugation이 일어나지 않았다. 또한 염색체의 질소고정 변이주와 plasmid 질소고정 변이주의 유전적 특성을 조사하였다. 한편 gene fusion 및 nif 유전자 조절에 관한 연구로 ΦMud1 과 Mucts62를 이용하여 얻어진 nif-lac fused 변이주는 NH₄+ 전재하에서 현저한 β-galactosidase 활성도 감소를 보였다. 대두접종제 개발을 위한 기초 연구로 근류균주를 수집 유수균주를 선별하였다. 수도작 관련 질소고정에 관한 기초 연구로 pBR322 증폭에 관련된 조건 확립 및 E.coli와의 형질전환 각종조건을 확립하였다. 기타 유전자 조작에 의한 dehydrogenase 다량생산에 관한 연구가 실시되었다.

Abstract ▼

1. Basic research for genetic study of nitrogen fixing bacteria K. pneumoniae M5aℓ fixed dinitrogen on NFM and SN media, and conjugation frequency of pRD 1 in E. coli to E. coli Kl2 was 5.6×$10^{-2}$, to K. pneumoniae M5aℓ was 5.0x$10^{-6}$, but to Rhizobium sp. was near zero.

1. Basic research for genetic study of nitrogen fixing bacteria K. pneumoniae M5aℓ fixed dinitrogen on NFM and SN media, and conjugation frequency of pRD 1 in E. coli to E. coli Kl2 was 5.6×$10^{-2}$, to K. pneumoniae M5aℓ was 5.0x$10^{-6}$, but to Rhizobium sp. was near zero. Good growth of over 60% of Rhizobium sp. on LN medium suggested that the possibility of the genetic study. Transduction frequencies of his and nif genes by ${phi}$P1vir. were very low or near zero.2. Genetic complementation test 18 chromosomal nif mutants and 16 plasmid nif mutants were used in complementation test by plate method, and the conjugation frequencies of UN series, CK series, UNF 4400, and UNF 4028 were very low, but those of the other UNF series and PC series were high.3. Gene fusion and studies on the regulation of nitrogen fixation genes Nif-lac gene fused mutants were selected using transposon ${phi}$Mudl and Mucts62. Complementation tests were carried out to characterize genetic markers with 27 mutants which grew well, and finally LX-9 and LX-22 were used for the nif gene regulation study due to their reliable genetic markers. All nif-lac gene fused mutants showed significant decrease of β-galactosidase activities by addition of $NH_4^{+}$. Nif structural gene induction of LX-9 and LX-22 were increased rapidly in initial 4 hrs and slowly after that time. The strong repression effects of nif genes were observed in the presence of $NH_4^{+}$, urea, $NO_3^{-}$, serine, glutamine, asparagine but were very weak in histidine, glutumic acid, arginine , casamino acid.4. Development of legume inoculant 370 strains of Rhizobia were isolated from about 100 regions in Korea, and the host of 172 strains among them was soybean. They were tested in infectivity to host and efficiency of nitrogen fixation and highly effective strains R-97, R-214, R-224, R-240, R-278 were selected. It would be expected that they would be used as superior legume inoculants for their actylene reducing activities (14.0-16.7 μmole $C_2H_4$/g nodule·hr) were higher than that of R. japonicum L-259 from USA and increased host dry matter by 1.3-1.6 times compared with control. In the future, larger culture system of legume inoculant will be established and through the strain development and field test, they will be contributed to the increase of soybean production.5. Rhizospheric nitrogen fixation in rice fied Plasmid pBR 322 in E. coli GM4 was amplified upon addition of 50-150 μg/ml chloramphenicol to log phase culture and 1 mg pBR 322 was purified from 3.5 ℓ culture of E. coli GM4. High transformation efficiency was observed when pBR 322 DNA was added at the ratio of 1.0x$10^7$ CFU/mg plasmid DNA to log phase E. coli C600 pretreated with 5 mM $MgC1_2$, 100 mM $CaC1_2$at pH 7.0 for 15 min, and transformation efficiency was 0.8-1.0x$10^6$ transformants/μg pBR 322 DNA. In parallel, rhizospheric nitrogen fixing bacteria were isolated from paddy soil and acetylene reducing activities were assayed and related plasmids were detected.6. Gene cloning for hyperproduction of dehydrogenase Transformation efficiency was 5x$10^6$/μg pBR 322 when recombinant DNA obtained with restriction enzyme Pst 1 was transformed into E. coli LE 392. Among transformants $tet^R$ colonies were about 3×$10^4$ but $tet^R$, $amp^S$ colony was not obtained. 700 μg chromosomal DNA was obtained from 1.7 g Desulfobivrio desulfurican and 3-10 kb DNA fragments were produced upon treatment of restriction enzyme EcoRl and pBR 322 treated with phosphatase was not self-ligated.

목차 Contents

• 표지...1
• 제1장 서론...15
• 제2장 실험재료 및 방법...19
• 제1절 사용균주...19
• 제2절 배지조성 및 시약조성...20
• 제3절 Conjugation 및 genetic complementation test...23
• 제4절 Transduction with ${\phi}Plkc$...24
• 제5절 ${\phi}Mud1$ 및 Mucts62 hp15 preparation 및 gene fusion...26
• 제6절 Rhizobium의 분리...29
• 제7절 효율적인 Rhizobium japonicum의 선별...30
• 제3장 실험결과 및 고찰...35
• 제1절 질소고정세균의 유전적 연구를 위한 기본연구...35
• 1. K.pneumoniae $M5\;a\ell$ 의 배양상 특성...35
• 2. pRD1 plasmid의 conjugation transfer...35
• 3. Rhizobium japonicum의 LN 배지에서의 생장...36
• 4. Transduction of nif genes with ${\phi}P1$ virus...36
• 제2절 Genetic complementation test...37
• 제3절 Gene fusion 및 nif 유전자의 regulation연구...39
• 1. nif-lac fused변이주 선별...39
• 2. $\beta$-galactosidase induction...40
• 3. 여러가지 질소원의 nif genes에 대한 repression효과...42
• 4. Nitrogenase의 autogeneous regulation...44
• 제4절 Rhizobium의 분리...45
• 제5절 Nodule형성능(infectivity)및 질소고정능(effectiveness)시험...46
• 제4장 결론 및 건의사항...50
• 참고문헌...54
• 부록1. 수도작 관련 질소고정에 관한 연구...56