摘要
利用转座子Tn5对质粒pJB-B6既定位诱变，经同源交换，筛选获得一株Rhizobiumhuakuii107胞外糖合成缺陷（Exo－）变种RH983。三亲本杂交实验显示，pJB-B6及其亚克隆pJB-B601均可纠正变种RH983的胞外多糖合成缺陷。pJB-B601的2．3kbDNA片段的核苷酸顺序表明，该片段内存在一个完整的开放阅读框架（ORF）。ORF全长1170bp，编码390个氨基酸的蛋白，该蛋白与Rhi－zobiummeliloti的糖基转移酶ExoL有56．7％．的同源性，称为RhexoL。利用启动子探测质粒，构建了RhexoL-lacZ转录融合子，发现RhexoL基因5’上游有较强的启动子活性。
Recombinant plasmid pJB-B6 was mutagenized by Tn5-region-directed insertion. By conjugation of the mutagenized plasmid into Rhizobium huakuii 107 containing a P-group plamid which is incompatible with pJB-B6, one R.hukuii 107 EPS-deficient(Exo- ) mutant Rh983 was isolated. From pJB-B6 which could complement the Exo- mutant, a 2-3 b Pst I -Sacr I fragment was subcloned in pRK415 (Fig- 1 ). The restulting plasmid pJB-B601 could restore the Exo- phenotype of Rh983. Determination of the complete nucleotide sequence of the fragment (Fig. 2) showed that it contained the structural gene of a glucosyL-transferase , as well as the 5, - and 3 '- flanking regions. An open reading frame of 1 170 base pairs was identified as R. huakuii exoL gene (Fig. 2). The MW of ExoL, as deduced from the nucleotide sequence, was estimated at 44 kD. Comparison of the nucleotide sequences revealed a high similarity between the exoL genes of R. huakuii and of R. meliloti (Fig. 3 ). The deduced amino acid sequence of R. huakuii ExoL also showed a high similarity with that of R. meliloti ExoL. Furthermore , the exoL-lacZ transcription fusion was constructed and the expression of exoL -lacZ was analzed.
基金
国家攀登计划资助
