Functional Genes Screened From A Transposon-inserted Mutant Library Of A Dietzia Strain

The oil leakage caused by each link from the exploitation of oil to the final use has caused serious environmental pollution.The techniques of microbial remediation is the most promising method for the treatment of oil environmental pollution.Mastering the alkane degradation mechanism of the oil-degrading strain is the key to the construction of genetic engineering bacteria and the utilization of this technology in the later period.Dietzia bacteria can degrade a variety of n-alkanes,and grow at high salinity and alkalinity.It is important to construct a robust mutant library to lay the foundation for the further research of the molecular mechanisms of n-alkane degradation of Dietzia bacteria,as well as its salt and alkali tolerance.We set strain Dietzia sp.DQ12-45-1b as a model and used a dual plasmid system of pTip-ist AB-sacB and p RTSK-sac B to construct the transposon-inserted mutant library.We successfully constructed a transposon-inserted mutant library with a storage capacity of 30,720.Southern blot analysis of the random selected mutants showed that these mutants were all single insertion mutations.After more than 20-time passages of these mutants,the inserted sequences were still remained in their chromosomes,suggesting the mutant library robust for target gene screening.We designed a high-throughput gene screening method for n-hexadecane degradation.After multiple screening of MF solid medium,MF solid medium and MF liquid medium,seven degradation genes related to n-hexadecane were successfully screened out of Dietzia sp.DQ12-45-1b: Contig3-orf00026(DUF4263 domain-containing protein),contig8-orf00015(Hypothetical protein protein),contig8-orf00068(Ferredoxin),contig8-orf00071(Alkane Hydroxylase CYP153),contig8-orf00074(Ferredoxin reductase),contig46-orf01661(ABC transporter ATP-binding protein),contig46-orf04630(Hypothetical protein protein).The knockout strains were obtained by knocking out target genes on the genome,and the experients of growth curve and the degradation rates of n-hexadecane were measured.The results showed that the degradation rates of the DQ12-45-1b wild-type strain was 91.17 ± 4.70%.The rates of DQ12-45-1b wild type strain has a statistically significant difference(P < 0.05)with ?3-26,?8-15,?8-68,?8-71,?8-74,?46-1661,?46-4630.About the knockout strains,the highest degradation rate was 83.70 ± 6.10%,the lowest rates was 15.92 ± 1.82%.The results showed that the knockout genes of these seven knockout strains were all related to the degradation and utilization of nhexadecane.The complemenation strains were obtained by covering target genes Contig8-orf00068,Contig8-orf00071,Contig8-orf00074 to the DQ12-45-1b wild-type strain,and the experiments of growth curves and the degradation rates of n-hexadecane were measured.The results showed that the core of the degradation system was not the alkane hydroxylase CYP153,but the ferredoxin protein responsible for electron transfer,and it may play an extremely important role in the degradation of alkane by the strain.