大豆和花生根瘤菌氢酶的研究  

Studies on Hydrogenase from Rhizobium japonicum and Rhizobium arachis

作　　者：许良树[1] 张凤章[1] 龙敏南[1] 曾定[1] 黄河清[1] 刘月英[1] 刘广发[1] 

机构地区：[1]厦门大学生命科学学院,福建厦门361005

出　　处：《厦门大学学报（自然科学版）》2001年第2期604-610,共7页Journal of Xiamen University：Natural Science

基　　金：国家自然科学基金!(39370 0 6 2 ;39770 0 70 );福建省自然科学基金!(B9910 0 0 1)资助项目

摘　　要：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 4 0 %～ 6 0 % .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 Sephacryl S- 2 0 0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 (6 0 k D,6 5k D)、小 (30 k D,35k D)两个亚基 ;均为 Ni Fe-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 p Z- 55.利用多种限制性内切酶构建了质粒 p Z- 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 % .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 wit

关 键 词：大豆根瘤菌 花生根瘤菌 吸氢酶 吸氢酶基因 吸氢基因转移 固氮效率 

分 类 号：Q939.114[生物学—微生物学] Q946.549