| Summary: | 碩士 === 東吳大學 === 微生物學系 === 95 === In aerobic gram positive bacteria, phthalate is metabolized by dioxygenase-initiated pathway. Researchers have shown that the α-subunit of the phthalate dioxygenase is consisted of phtA and other related genes. In the present study, the diversity of phtA gene in environmental samples was assayed by PCR-denaturing gradient gel electrophoresis (DGGE) method using a newly designed primer set ( phtA F189 [5’-STTYAAYATGTGCCTS CAYC-3’] and phtA R685 [5’-GTTATGGACGAAGCTCATGT-3’] ). Of the 17 bacterial strains isolated from phthalates polluted environments, phtA genes were detected in 10 dibutyl phthalate (DBP) degraders. Further analysis indicated that all the amplified sequences shown the highest similarity to α-subunit of the phthalate dioxygenase of gram positive bacteria listed in the NCBI GeneBank. From these results, we have concluded that primer set developed in the present can effectively amplify the α-subunit of the phthalate dioxygenase. Molecular identification (sequence comparison of 16S rDNA) has shown that all the environmental isolates belong to Actinomycetales. Degradation experiments have shown that out of 8 DBP isolates only 6 shown the abilities to degrade DEHP (Di-(2-ethylhexyl) Phthalate). The diversity of phtA genes was examined in DNA extracts from different soil environments located at various parts of Taiwan and from previously isolated phthalate degraders. Differences in DGGE patterns were observed between each environmental sample, indicated that the diversity of phtA analogues in the environment is larger than those observed from described phtA analogues of the tested isolates. The results presented here have showed that the primer set phtA F189/ R685 can not only effectively reveal the diversity of phtA gene in environmental samples but also provide a resource for future biodegradation, and functional gene diversity analyses.
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