大豆和花生根瘤菌氢酶的研究

根瘤菌在共生固氮过程中因放H2所消耗的能量约占固所氮总能量的40%-6%。吸氢酶则能回收和利用固氮过程所放的H2，养活能量损失，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Sephacry S-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（60kD,65kD)、小（30kD,35kD)两个亚基，均为NiFe-氢酶，并且有较高的吸H2活性。大豆根瘤菌氢酶的纯酶组分不含Cyt b599。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%。

Studies on Hydrogenase from Rhizobium japonicum and Rhizobium arachis

Hydrogen produced by nitrogenase consumed 40~60% of energy of symbiotic nitrogen fixation. Hydrogenase can uptake and reuse the H 2 produced by nitrogenase, which results in decreasing the loss of energy and increasing the efficiency of nitrogen fixation. The membrane bound 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 H 2 and CO 2. It is evident that H 2 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 H 2 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 H 2 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% and 8.9% respectively. The peanut yield inoculated with transconjugant increased by 9.6% in comparison with that inoculated with recipient.