浑球红细菌Rhodobacter　sphaeroides吸氨酶(hup)基因簇的分离与鉴定  

Isolation and Identification of hup Gene Cluster from Rhodobacter sphaeroides

作　　者：梁建光[1] 吴永强[1] 宋鸿遇[1] AnnetteColbeau PauletteM.Vignais 

机构地区：[1]中国科学院上海植物生理研究所,上海200032 [2]LaboratoiredeBiochimieMicrobiennne

出　　处：《植物生理学报（0257-4829)》1998年第3期233-239,共7页Acta Phytophysiologica Sinica

基　　金：国家高技术计划资助!863

摘　　要：以Rhodobactercapsulatus的hopS’L基因DNA片段为探针，通过Southem杂交，从构建的Rhodobactersphaeroides601基因库中调取hup基因。阳性克隆Cosmid1可与Hup突变株JP91（HupS－）、RCC8（HupR-）以及BSE8（HupT-）互补，而Cosmid3和Cosmid9只能与BSE8互补；试验所产生的接合转移子均恢复了吸氢酶的活性和自养生长能力、将Cosmidl的3．5kbPstⅠ和4．5kbBamHⅠ片段分别亚克隆到pWY11和pWY10中。pWY11和pWY10的部分DNA序列与R．capsulatus中的相关基因具有很高的同源性，从而证明了所得到的Cosmid1确实含有R．sphaeroides的基因簇。Three cosmids, Cosmid1,Cosmid3 and Cosmid9, contalning hupand hyp genes of Rhodebacter sphaeroides were identified from a cosmidgene library of R. sphaeroides strain 601by hybridization with a 3. 5 An Hind Ⅲhant of Rhodobacter capsulatus hup gens . Cosmid1 restond the hydrogenaseactivity and autrophic growth of the Hup mutants JP91 (HuPS ) andRCC8 (HupR ) of R. capsulatus, and also restored the repression of hydroge expression in R. copsulatus HupT mutant BSES. Cosmid3 and Cosmidg were able to complement mutant BSE8 in vivo, but not mutant JP91 or RCC8 (Tables 2 and 3). This indicated that the 28. 8 An insert region of R. sphaeroides DNA in Cosmid1 (Fig. 1) carried a hup gene cluster including the hupR, hupS and hupT genes. A 3. 5 kb Pst I and a 4. 5 kbBamH I fragments were subcloned in pUC18 onto PWY11 and PWY10 respectively. PWY11 was shoWn to be homologous to the 1. 44 kb Eco RI fragment ofR. capsulatus hup R gene from plasmidpAC63. The nucleotide (nt) sequencing of 3' end of hupR gene of R. sphaeroide in PWY11 was obtained. The end of hupR was ended nt 264, then hypC gene was from nt 266 to nt 556, the hyp D gene began at nt 553 (Fig. 2). The nt Sequences of hypC and hypD were linked by ATGA. Thecomparisons of nucleotides and deduced amino acid sequence indicated that the Predicted HrpC Protein of R. sphaeroides contained 96 amino acids and its amino acid sequence showed 76% identity with the HypC of R.cap sulatus 1 and the 3' terminal of hup R'showed 81. 9% identity between hup Rgenes of R. sphaeroides and R. capsulatus. In E. coli, the Product of hypC gene appears to adjust the activity of hydrogenase 3, which transfers electrons to protons and releases hydrogen. Homologies of the R. capsulatus hypC gene were found in R. sphaeroides but the function of its product has not yet been investigated. The nucleotide sequence of pWY10obtained showed 82. 9% identity with hupL of R. capsulatus from nt 1943 to nt 2617 (Fig. 3) and the deduced amino acid sequence showed 84. 1% identity with HupL of R

关 键 词：浑球红细菌 吸氢酶 基因簇 hup基因 

分 类 号：Q781[生物学—分子生物学] Q74