含花生根瘤菌吸氢基因重组质粒pZ-55的特性分析

Characterization Analysis of Recombinant Plasmid pZ-55 Containing hup Genes from Rhizobium arachis

应用含豌豆根瘤菌部分吸氢基因的探针及ＰＣＲ分析，从花生根瘤菌基因文库中筛选到含有吸氢基因的重组质粒ｐＺ５５。用ＢａｍＨⅠ、ＥｃｏＲⅠ和ＫｐｎⅠ等内切酶对ｐＺ５５进行酶切分析，构建了ｐＺ５５的酶切物理图谱。经三亲本杂交分析，ｐＺ５５能互补诱变株Ｌｎ１（Ｈｕｐ－），获得在自生条件下表达吸氢活性。

In order to obtain the DNA fragments of the hup structural genes conveniently and quantitatively, part of hup structural genes from pAL618 (a recombinant containing hup genes of Rhizobium leguminosarum ) was sucessfully subcolned in E.coli DH5α by using plasmid pBluescriptⅡKS(+) as vector. The fragment of the hup structural genes was isolated from the recombinant plasmid pBlue 6.0 and was labeled with Dig 11 dUTP by random primers. Two primers of PCR were synthesized according to nucleotide sequence of hup structural genes ( hup S and hup L) of Rhizobium japonicum and Rhizobium leguminosarum . One recombinant cosmid of pZ 55 was isolated from 71 colonies of the gene library by PCR screening and Dig probe hybridization (Figs.1,2), which possibly contained hup genes. The size of insert foreign fragment in pZ 55 was about 19.6 kb. Bam HⅠ, Eco RⅠand Kpn Ⅰwere used in mapping analysis to indentify the physical organization of the insert foreign fragment(Fig.3). The cosmid of pZ 55 was transferred from E.coli strains to the Hup - recipients: Ln 1 (a mutant of R. arachis derived from strain L8 3) by triparental mating using the plasmid pRK2013 as the helper plasmid. According to the analysis of plasmid DNA, the hybrid cosmids had been transconjugated into the recipients. The transconjugant EL 55 showed high level of H 2 uptake activity when induced under free living microaerobic condictions (Fig.4).