野生大豆(Glycine soja SH1)球蛋白glycinin Gy4基因家族的两种表达拷贝

本文研究我国野生大豆(Glycine soja)种子贮藏蛋白基因的结构,并与栽培大豆进行比较,构建成野生大豆Glycine soja SH1和栽培大豆Glycine max子叶cDNA库。用已知含球蛋白glycinin Gy 4基因的克隆DNA λS 312为探针,从两个cDNA库中分离出6个克隆。其中两个克隆pWS 228与pWS 242含全长cDNA,克隆cDNA的限制酶图谱和cDNA与基因组DNA的Southern法杂交表明它们代表野生大豆球蛋白glycinin Gy 4基因家族的两种表达拷贝。pWS 228 cDNA的部分序列已测定并与栽培大豆相应顺序作比较。分子杂交还证明Ⅱ类glycinin基因家族的组编在野生大豆胚形成过程中的变动。     

EFFECT OF nifA PRODUCT ON SUPPRESSION OF Nif~- PHENOTYPE OF gin MUTATION AND CONSTITUTIVE SYNTHESIS OF NITROGENASE IN Klebsiella pneumoniae

This paper describes the role of nifA product on the ammonia regulation of nitrogen fixation in K. pneumoniae. A plasmid carrying nifA gene under the promoter of tetracycline resistance gene was constructed. When this nifA carrying plasmid was introduced into a glnAG mutant, the Nif~-phenotype of this gln mutant was suppressed. Furthermore, when the plasmid was introduced into the wild type and glnAG mutant, derepression of nitrogenase synthesis in ammonia occurred in both strains and the products of nif genes can be detected by two-dimensional gel electrophoresis in the extracts of these ammonia-grown bacterial cells.The constitutive synthesis of nitrogenase in NH_4~ was also demonstrated in free living nitrogen-fixing bacteria, Enterobacter cloacae, when the bacteria received the plasmid carrying nifA gent from K. pneumoniae.     

基因nifA产物对肺炎克氏杆菌(Klebsiella pneumoniae)gln突变型的Nif~-表型的校正和固氮酶的组成型合成的作用

本文叙述固氮基因nifA对肺炎克氏杆菌(Klebsiella pneumoniae)固氮作用的氨调节的影响。首先建构一个带有基因nifA的重组质粒,使基因nifA处于四环素抗性基因启动子控制之下。当这个建构成的带有基因nifA的质粒引入谷氨酰胺合成酶基因glnAG的突变型后,这个突变型的Nif~-表型就得到校正。此外,当这个质粒引入野生型和gln突变型后,两者在氨存在下都发生固氮酶合成的去阻遏作用,并在双向凝胶电泳上可以检测出固氮基因产物的存在。同样地,当自生固氮菌阴沟肠杆菌(Enterobacter cloacae)E26接受了这个重组质粒后也能观察到固氮酶的组成型合成。     

STUDIES ON HETEROLOGOUS EXPRESSION OF Rhizobium meliloti nifA GENE AND OXYGEN SENSITIVITY OF ITS PRODUCT

When the R. meliloti (Rm) nifA'-lacZ fusion-carrying plasmids were introduced intothe strains of Agrobacterium tumefaciens, R. trifolii and R. astragalus, β-galactosidaseactivity was demonstrated. However, activity was not induced by microaerobiosis. Further-more, R. meliloti nifA'-lacZ fusion was also not expressed in the nodule bacteroids ofR. trifolii and R. astragalus. We speculate that some factor(s) important for the inductionof Rm nifA presumed to be the fixLJ regulatory system would not be operative in thesebacteria. Experiments using R. meliloti nifH'-lacZ/ K. Pneumoniae nifH'-lacZ fusion and theconstitutive Rm nifA system to test the nifA-dependent expression of nifH'-lacZ underaerobic and microaerobic conditions in E. coli were performed. The inhibition of the RmnifA activation of nifH'-lacZ expression in the bacteria grown in the aerobic conditionwas shown. Assays on the Rm nifA-m RNA produced by the constitutive Rm nifA in E.coli under aerobic and microaerobic conditions with the cloned ni     

TWO EXPRESSED COPIES OF GLYCININ Gy4 GENE SUBFAMILY IN WILD SOYBEAN Glycine soja SHI

To study the structure of genes encoding seed storage proteins of the wild variety of soybean (Glycine soja) in comparison with those of the cultivated (Glycine max),cDNA libraries from cotyledon poly(A)~+ RNA of both wild and cultivated soybeans were constructed.Six clones were isolated using the genomic clone λS312 which contains the coding sequence for the glycinin subunit precursor (A_5A_4B_3) as probe for molecular hybridization. Two clones pWS228 and pWS242 contain the full length of cDNA.Restriction mapping of the cloned cDNA and Southern hybridization of the cDNA with genomic DNA showed two expressed copies of glycinin Gy4 gene subfamily in wild soybean Glycine soja SHI. Alteration of DNA arrangement in group-Ⅱ glycinin gene subfamily during embryogenesis was also demonstrated.     

肺炎克氏杆菌(Klebsiella pneumoniae)his D非连锁性Nif突变型的遗传定位和生化特性

两个hisD非连锁性基因NifC5和NifC7已在肺炎克氏杆菌(K.pneumoniae)染色体上进行定位.NifC5和NifC7的顺序是:NifC5-gltB-NifC7-argG.噬菌体P1感染的大肠杆菌(E.coli K12)溶菌液能够与突变型C-7进行转导而产生Nif⁺转导子,但与hisD连锁性nif突变型进行转导则得不到Nif~+转导子.这表明NifC7不是固氮系统的结构基因,但存在于大肠杆菌之中.许多实验证明NifC7是属于谷氨酸合成酶基因.SDS凝胶电泳分析表明固氮还原酶的含量明显减低.免疫电泳分析说明固氮酶和固氮还原酶的含量都有所减低.     

nifA基因对于Escherichia coli中受nif启动子控制的lacZ表达的作用

本文作者构建了两类重组质粒:一类包含nifH启动子-lacZ的融合,另一类包含了受质粒四环素抗性基因启动子控制的nifA基因。由于这两类质粒不属于同一的不亲和群,因而能够在Escherichia coli的同一细胞内各自复制。发现nifA基因产物对激活nif启动子的转录是必需的,并证实了nifA产物的温度敏感性,以及在nifA参与下nfi启动子控制lacZ的表达受到铵和L-丝氨酸的阻遏。     

肺炎克氏杆菌(Klebsiella pneumoniae)中固氮基因nifH,nifC和nifJ的特性和互补分析

为了阐明固氮系统中的固氮基因的特性,我们应用了不同固氮基因突变体的互补试验.以大肠杆菌(E.coliK12)Jc-5466的质体突变体PRD-nif~-转入肺炎克氏杆菌(K.pneumoniae)不同nif~-的受体菌中来构成Nif~-/Nif~-的杂合子.除了先前报道过的基因外,我们找到了一个在固氮系统中必要的新基因nifC.依据细菌噬菌体P₁的共转导和三点正反交试验的结果,nifC定位于nifH和nifJ之间.它们的排列顺序是his D,nif Q,nif B,nif A,nif L,nif F,nif M,nif V,nif S,nif U,nif N,nif E,nif K,nif D,nif H,nif C和nif J.从测定固氮基因的生化表型的试验指出,nif C可能是对固氮酶中的铁钼辅因子的合成或激活有关.nif H除编码固氮还原酶外,它的功能也与合成固氮酶有关.而nif J可能是nif K,nif D,nif H或nif F的表达所需要的.     

苜蓿根瘤菌多拷贝固氮基因启动子对根瘤发育的抑制

以荧光素酶基因作为报道基因研究苜蓿根瘤菌nif/fix基因在根瘤发育过程中的激活,发现多拷贝的nifHDK启动子和fixABCX启动子抑制根瘤的发育和固氮活性,与nifA突变型的表型相同.用β-半乳糖苷酶基因作为报道基因得到同样的结果。但是低拷贝的nifHDK启动子或fixABCX启动子对根瘤发育和固氮活性没有影响。固氮基因启动子的多拷贝抑制效应进一步说明nifA产物除了作为共生固氮基因转录的正调节因子外,对根瘤的发育也有作用。     

苜蓿根瘤菌(Rhizobium meliloti)nifA基因的异源表达及其产物的氧敏感性

当在土壤农杆菌、三叶草根瘤菌和紫云英根瘤菌中分别引入Rm nifA'-lacZ的质粒,可以测得β-gal活力,但其活力不受微氧诱导。当带有Rm nifA'-lacZ的三叶草根瘤菌和紫云英根瘤菌处于共生状态,β-gal的活力也无明显提高。推测苜蓿根瘤菌 nifA的诱导激活是受某些因子如fixLJ的专一作用。以大肠杆菌为材料在有氧和微氧下观察nifH'-lacZ受nifA的激活表达。RmnifA在有氧下不能激活Rm nifH'-lacZ和KpnifH'-lacZ的表达。同时证实RmnifA的mRNA在有氧下含量显著减少。