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Analysis of gene transfer and expression mechanisms in aquatic ecosystems using the Aquatron

使用 Aquatron 分析水生生态系统中的基因转移和表达机制

基本信息

批准号：
13309009
负责人：
KAWABATA Zen'ichiro
金额：
$ 34.36万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

Operational conditions for replicable aquatrons were clarified. Blooms of cyanobacterium Microcystis aeruginosa was induced artificially in the aquatrons. In these aquatrons, gene transfer of pT7GFP from E.coli (pT7GFP) to 0.1 to 0.8% of indigenous bacteria was detected within 3 to 5 days, by means of FISH targeted to pT7GFP couple with fluorescent antibody method. It was also found that extracellular metabolic products enhanced conjugal gene transfer of pBHR1 from E.coli S17-1(pBHR1) to Pseudomonas stutzeri.E.coli with a plasmid coding GFP gene was exposed to autolysis, infection of phage and predation by protozoa. Quantification of dissolved DNA, as gene source in a natural aquatic environments, in the treated sample, revealed that dissolved DNA from autolysis and infection of phage are likely to more contribute than the other to horizontal gene transfer among bacteria.Bacillus strains isolated from 17 various sites in the world possessed identical mer operon on similar transposons. This finding suggested that transposons may contribute the worldwide distribution and horizontal dissemination of the mer operons among Bacillus strains in natural environments. E.coli DH5α was incorporated with both a transposon, which was isolated from natural environment, on untransformable pGEM and transformable plasmid pR388. The transposon in the E.coli DH5α was transferred to E.coli S17-1 by conjugation. These experiments indicated that there existed transposition of the transposon from pGEM to pR388 in a cell.

阐明了可复制水族箱的运行条件。人工诱导水族箱中的铜绿微囊藻水华。利用荧光抗体方法对pT7GFP偶联进行FISH，在3~5天内检测到pT7GFP从大肠杆菌(PT7GFP)向0.1%~0.8%的原生菌的基因转移。研究还发现，胞外代谢产物促进了pBHR1从大肠杆菌S17-1(PBHR1)到斯氏假单胞菌的接合基因转移。携带GFP基因的大肠杆菌暴露于自溶、噬菌体感染和原虫的捕食。对自然水环境中作为基因源的溶解DNA的定量检测表明，自溶和噬菌体感染的溶解DNA可能对细菌之间的水平基因转移做出了更大的贡献。世界上17个不同地点的芽孢杆菌菌株在相似的转座子上具有相同的聚合子操纵子。这一发现表明，转座子可能有助于自然环境中芽孢杆菌操纵子在世界范围内的分布和水平传播。将从自然环境中分离到的转座子与不能转化的pGEM上的转座子和可转化的pR388质粒整合到E.coliDH5α上。通过接合的方法将E.ColiDH5α中的转座子转移到E.ColiS17-1中。这些实验表明，在细胞内存在转座子从pGEM到pR388的转座。