【作者】 毕洪凯；

【导师】 武波；

【作者基本信息】 广西大学 ， 微生物学， 2004， 硕士

【摘要】 本工作从长期受苯胺污染的废水排放口的活性污泥中筛选到一株高效苯胺降解细菌GXA7，能够在以苯胺为唯一碳源和氮源的无机盐培养基上生长。通过对菌株GXA7在不同温度、pH值，以及培养基中不同苯胺浓度条件下的生长和降解苯胺的情况，我们初步确定了GXA7的最适生长和降解苯胺的温度为32℃～36℃，最适pH值为7.0，苯胺最高的耐受浓度为2500mg／L。通过传统表型分类法及16SrDNA编码序列同源性比较分析，我们鉴定该菌株为Delftia sp．。通过设计苯胺双加氧酶基因特异引物，以菌株GXA7基因组DNA为模板，PCR扩增出两段与Acinetobacter sp．strain：YAA plasmid：pYA1 DNA（Accession number：D86080）苯胺双加氧酶基因分别有93％和98％的同源性片断。经Southern杂交验证，PCR扩增产物atd788和atd762可以作为筛选文库的探针。利用粘粒pLAFR3作为载体，制备基因组DNA部分酶切片断，经体外包装、转染E．coli EPl 100，在选择培养基平板上获得1万多个重组克隆子，构建了菌株GXA7的基因组粘粒文库。以经³²P标记的PCR扩增产物atd788作为探针，菌落原位杂交，得到五个可能的阳性克隆，然后经Southern blotting验证，转化子pGXA240及pGXA310为阳性克隆。亚克隆测序分析，我们初步确认克隆到苯胺双加氧酶基因簇。同时完成了Delftia sp. GXA7苯胺双加氧酶基因簇atdA1A2A3A4全序列的测定，并对其核苷酸及其推导的氨基酸序列进行分析。更多还原

【Abstract】 A bacterial strain GXA7, which was able to grow on aniline as a sole carbon, nitrogen and energy sources, was isolated from the activated sludge drained from the wastewater polluted by aniline for a long time. The optimum temperature and pH for its growth and aniline degradation were 32C~36C and 7.0, respectively. In suitable conditions strain GXA7 can degrade aniline with 2500mg/L at most. Based on phenotypic identification and 16S rDNA sequence analysis, strain GXA7 was identified as Delftia sp.. With specific primers designed, PCR products atd788 and atd762 were amplified by PCR. The two fragments were sequenced and closely related to aniline dioxygenase gene of Acinetobacter sp. (Accession number: D86080) with 93% and 98% overall similarity respectively. A genomic library of strain GXA7 was constructed in the cosmid vector pLAFR3 using E.coli EPIlOO as the host strain. Two recombinants, pGXA240 and pGXA310 were identified from the genomic library using in situ hybridization and Southern blotting probed with the PCR product atd788. By the analysis of subclone sequencing, we made certain that the aniline dioxygenase gene cluster of strain GXA7 was cloned.更多还原