CAJ | 학술논문

根瘤菌在共生固氮过程中因放H2所消耗的能量约占固氮总能量的40％～60％．吸氢酶则能回收和利用固氮过程所放的H2，减少能量损失，从而提高共生固氮效率．在厌氧条件下，加入防止酶蛋白聚合的试剂，利用DEAE-纤维素和SephacrylS-200柱层析，从自养性大豆根瘤菌和花生根瘤菌类菌体中分离并提纯膜结合态氢酶．纯化的两种氢酶表现相近的分子特征：均含有大(60 kD，65kD)、小(30 kD，35 kD)两个亚基；均为NiFe-氢酶，并具有较高的吸H2活性．大豆根瘤菌氢酶的纯酶组分不含Cyt b559．花生根瘤菌L8-3菌株能进行化能自养生长，诱导出高吸H2活性．根瘤菌的吸H2能明显提高固氮活性．从具有高吸H2活性的花生根瘤菌中分离并克隆吸氢基因，采用PCR和探针杂交技术，获得含有吸氢基因的质粒pZ-55．利用多种限制性内切酶构建了质粒pZ-55的物理图谱．通过三亲本杂交，将含吸氢基因的重组质粒转移到不吸H2的花生和毛豆根瘤菌中，所获得的结合株在自生和共生条件下均表达吸H2活性．以结合株接种大田花生，获得的共生根瘤的吸H2活性比接种受体株提高4倍，花生叶片和种子的含N量、产量分别提高1．7％、8．9％和9．6％．
근류균재공생고담과정중인방H2소소모적능량약점고담총능량적40％～60％．흡경매칙능회수화이용고담과정소방적H2，감소능량손실，종이제고공생고담효솔．재염양조건하，가입방지매단백취합적시제，이용DEAE-섬유소화SephacrylS-200주층석，종자양성대두근류균화화생근류균류균체중분리병제순막결합태경매．순화적량충경매표현상근적분자특정：균함유대(60 kD，65kD)、소(30 kD，35 kD)량개아기；균위NiFe-경매，병구유교고적흡H2활성．대두근류균경매적순매조분불함Cyt b559．화생근류균L8-3균주능진행화능자양생장，유도출고흡H2활성．근류균적흡H2능명현제고고담활성．종구유고흡H2활성적화생근류균중분리병극륭흡경기인，채용PCR화탐침잡교기술，획득함유흡경기인적질립pZ-55．이용다충한제성내절매구건료질립pZ-55적물리도보．통과삼친본잡교，장함흡경기인적중조질립전이도불흡H2적화생화모두근류균중，소획득적결합주재자생화공생조건하균표체흡H2활성．이결합주접충대전화생，획득적공생근류적흡H2활성비접충수체주제고4배，화생협편화충자적함N량、산량분별제고1．7％、8．9％화9．6％．
Hydrogen produced by nitrogenase consumed 40～60%0 of energy of symbiotic nitro gen-fixation. Hydrogenase can uptake and reuse the H2 produced by nitrogenase, which results in decreasing the loss of energy and increasing the efficiency of nitrogen fixation. The membranebound hydrogenase from autotrophical cultured Rhizobium japanicum and from the bacteroids of peanut nodule have been purified and characterized. The hydrogenase from R. japonicum consists of two subunits (60 kD, 30 kD). The molecular weight of large and small subunits of hydrogenase from R. arachis is about 65 kD and 35 kD. Both hydrogenases are NiFe-hydrogenase. No cytochrome b(559) could be detected in the fractions with the highest specific activity of hydrogenase from R. japonicum. R. arachis L8-3 strains(hup+) expressed high hydrogenase activity when grow autotrophicully in mineral salt-vitamins medium and gas mixture containing H2 and CO2. It is evident that H2 uptake by hydrogenase significantly increased the dinitrogen fixation either in free-living cultures or in symbiotic nodules. A genomic library of R. arachis L8-3(hup+) has been constructed. Four clones(pZ-27, pZ-55, pZ-60, pZ-61) containing hup gene were screened by PCR and hydrogenase probe. The fragment of insert DNA of recombinant cosmid pZ-55 was about 19. 6 kb. When the cosmid pZ-55 was transferred into the hup- strain of R. arachis and hup- strain of R. japonicum by triparental matting, the transconjugants showed high levels of H2 uptake activities in free-living state. Inoculating test was carried out in the field by using the transconjugant containing pZ-55. The result indicated that H2-uptake activity of nodules inoculated with hup+ transconjugant was 4 fold high than those of nodule inoculated with recipient (hup-). The total nitrogen content of peanut leaves and seed increased by 17%0 and8.9 % respectively. The peanut yield inoculated with transconjugant increased by 9.6 %0 in comparison with that inoculated with recipient.