Genetic Transformation Of Polycyclic Aromatic Hydrocarbon Degrading Bacteria And Microorganism Repair Petroleum-contaminated Soil

With the development of the industry and increasing consumption of oil and other fossilfuels, the pollution of the environment becomes more and more severe. As one of the maincontaminations, polycyclic aromatic hydrocarbon has the largest virulence for organisms. Themost feasible method to eliminate the polycyclic aromatic hydrocarbon from the environmentis microbiological deterioration. This research cloned the coding genes of Ring-hydroxylatingdioxygenase large-subunit from Sphingobacterium multivorum SWH-2preserved in thelaboratory which can efficiently degrade polycyclic aromatic hydrocarbon and then proceededsequence. By means of the genetic engineering technology, this research converted thecoding genes into the chromosome DNA of SWH-2, finished genetic modification for SWH-2and got a new bacterial strain SWH-21. It has been proved by the degradation experiment thatSWH-21has higher capacity than SWH-2to degrade polycyclic aromatic hydrocarbon. Thisresearch also studied the remediation of the petroleum-contaminated soil under the effect ofSWH-21.The main research results are as follows.1． According to the coding gene sequence of Ring-hydroxylating dioxygenaselarge-subunit already existed on GeneBank, this research devised a pair of degenerate primer.According to the Tm value and GC content of the degenerate primer, this research chose theappropriate PCR conditions to proceed PRC, successfully amplified the coding gene sequenceof Ring-hydroxylating dioxygenase large-subunit and complete transformation afterconnecting the coding gene to carrier pMD-18.2． This research connected coding genes of Ring-hydroxylating dioxygenaselarge-subunit with suicide plasmid put-km1, constructed recombination suicide plasmid andsuccessfully transformed into esherichia coli DH5α—λpir.3．This research successfully integrated coding genes of Ring-hydroxylating dioxygenaselarge-subunit into the genomes of SWH-2by Triparental mating technology, finished geneticmodification for SWH-2and got a new genetic engineering bacterial strain SWH-21. Thisresearch also proceeded the comparison and analysis on degradation capability forphenanthrene and oil between SWH-21with SWH-2and reached a conclusion that the degradation rate of SWH-21is up to64.32%in10days,22.86%higher than SWH-2.4． This research applied SWH-21and SWH-2on the remediation ofpetroleum-contaminated soil. By proceeding comparison and analysis on the remediationcircumstances of the two bacteria, the results showed that the restoring capacity of SWH-21isclose to the capacity of SWH-2.