含吸氢酶基因（hup）嵌合质粒的构建和吸氢酶活性在阴沟肠杆菌中的表达  

The Construction of Chimeric Plasmid Carrying hup Genes from Bradyrhizobium japonicum and Expression of H_2-uptake Hydrogenase Activity in Enterobacter cloacae

作　　者：吴永强[1] 朱美珍[1] 梁建光[1] 华征[1] 宋鸿遇[1] 

机构地区：[1]中国科学院上海植物生理研究所

出　　处：《植物生理学报（0257-4829)》1996年第3期209-217,共9页Acta Phytophysiologica Sinica

基　　金：863计划资助

摘　　要：以质粒ｐＲＫ４０４为载体亚克隆含大豆根瘤菌吸氢酶结构基因（ｈｕｐ）的片段，构建成嵌合质粒ｐＨＲ１１、ｐＨＲ４和ｐＨＲ１０。通过三亲本杂交将这些嵌合质粒导入无吸氢活性的Ｒｈｏｄｏｂａｃｔｅｒｓｐｈａｅｒｏｉｄｅｓ２４１菌株（Ｈｕｐ－），均获得Ｈｕｐ＋的接合子。利用启动子检测质粒ｐＭＰ２２０证明，在ｈｕｐ对结构基因上游１．２ｋｂ内存在ｈｕｐ启动基因片段。以ｐＲＫ２０１３为助质粒可将ｐＨＲ１１导入Ｅｎｔｅｒｏｂａｃｔｅｒｃｌｏａｃａｅ和Ｋｌｅｂｓｉｅｌｌａｏｘｙｔｏｃａ等土壤固氮菌株。本文以接合子Ｅ．ｃｌｏａｃａｅＥＨ１１１３为例，通过对基因组ＤＮＡＳｏｕｔｈｅｒｎ杂交分析证明，嵌合质粒ｐＨＲ１１在ＥＨ１１１３中稳定贮存和复制。Ｈ２诱导接合子ＥＨ１１１３吸氢酶活性高表达，吸氢活性与放氢活性比值约为对照的两倍。当以延胡索酸为电子受体时，吸氢酶的吸氢作用支持菌株固氮酶活性的提高。hup structural genes, hupSL, wereisolated from B. japonicum cosmidpHU52 and subcloned downstream fromthe promoter of lacZ in vector plasmidPRK404, resulting in three chimericplasmids pHR11, pHR4 and pHR10:pHR11 conjoining 5. 9-kb Hind Ⅲ fragment with the hupSL genes in the positive orientation, pHR4 conjoining thesame size of the fragment with thehupSL genes in the opposite orientation,and pHR10 conjoining 8. 8-kb Hind Ⅲfragment with the hupSL genes in thepositive orientation (Fig. 1 ). When thethree subclones were conjugated respectively into the Hup-wild type strain ofR. sphaeroides 241, the resultingtransconjugantS were converted toHup+ phenotype as detected by HZ--uptake hydrogenase activity. To monitorthe activity of hUb promoter, hUb--buzZfusion was created by using promoterdetected plasmid pMP220(Fig. 2). TheresultS indicated that the expression ofp--galactosidase activity depended on theexpression of the hUb promoter within1. 2--kb fragment upstream hUbaL genesin the fusion.pHRI I was mobilized into somestrains of E. c￣, K￣ ￣,and ￣ sphaerde by triparentalmating at the frequency of 1 0--5 -- 1 0--6(Table 2). Southern hybridization usingacaL genes from B. oppeham and R.capons as probes revealed that the hUpstructural genes encoding HZ--uptake hydrogenase were localized in transconjugant E. (.la.ae EHI 113 on the 5. 9--kbHind W fragment of chimeric plasmidpHRI 1 and the DNA sequence of hULLgenes from B. modicum were homologous to a large extent with that of R.ac￣ hUbaL genes (Figs. 3, 4 ). Ahigh level of HZ--uptake hydrogenase activity was produced in E. cEHI 1 13 relative to E. c￣ wild type(Fig. 5). The ratio of H,--uptake activity to Hi evolution activity of EHI 1 13was increased to about twice that of thewild type (Table 3 ). An increase inHi--uptake hydrogenase and nitrogenaseactivities caused by the presence of10% Hi concentration in gas phase wasobserved in EHI 1 13 cells. Pretreatmentwith 40% acetylene, an inhibitor ofH,--uptake hydrogenase, caused a decreaSe in the activity of HZ--up

关 键 词：固氮细菌 吸氢酶基因 南粒 吸氢酶 阴沟肠杆菌 

分 类 号：Q945.13[生物学—植物学]