Construction Of Aquaculture Water Environmental Microbial Metagenomic Library And Screening Of Nitrite Degradation Clone

Metagenomics, also known as environmental microbial genomes or metagenome, is thetotal DNA of all tiny creatures in the environment (including bacteria and fungi). Microbialdiversity in natural environments is very rich, however, more than99%of microorganisms inenvironments are unculturable by using current techniques or in laboratory conditions. Theapplications of metagenomic methods to study the vast unknown microbes and microbialgenes will develop the knowledge of microbiology. Nitrite pollution in aquaculture water isworsening, and endangering the aquatic organisms growth and aquatic food safety. Screeningnitrite-degrading enzyme gene in aquaculture water using metagenomic technology isconducive to understand the denitrification process in this environment, and provide atheoretical basis for the nitrite pollution control.In this paper, the metagenomic library was construted on the aquaculture water samples.After purify DNA from the desired source, shearingDNA to approximately fragments,end-repairing the sheared DNA to blunt5’-phosphorylated ends,isolating the desired sizerange of end-repaired DNA, Ligating DNA to the vector, packaging the ligated DNA andplating on EPI300-T1R plating cells, a microbial metagenomic library was constructed.Thengenetic stability and library coverage of metagenomic library were tested. Fosmid clones werescreened by using the BTB media, in which nitrite as the sole nitrogen source and pHindicated by bromothymol blue (BTB). A clone was picked and inoculated in liquid BTBmedium, and fostered in37℃shaker. The medium pH and nitrite, nitrate, ammoniaconcentration were measured every12hours.The results showed that the clones coulddecompose more than50%nitrite in96hours, while the PH was maintained at about8.6.Meanwhile, we explored a new method to make a rapid identification of the inserted genefosmid library clones. The method based on semi-random primers temperature gradientPCR,and the front fragment and the terminal fragment of inserted gene could be amplificatedand identified. DNA band F/1-1(~1700bp) and F/1-2(~1300bp) were isolated and sequencedby using pCC2Forward Primer. The analysis suggested F/1-1and F/1-2DNA sequenceswere closely related to Acinetobacter calcoaceticus PHEA-2(CP002177.1) with a similarityof97%. Four DNA bands with different DNA sizes were obtained by using primer pair pCC2Reverse Primer and random primer. DNA band R/1-2(~1400bp) was isolated andsequenced by using pCC2Reverse Primer. The analysis suggested R/1-2DNA sequencewas also closely related to Acinetobacter calcoaceticus PHEA-2with a similarity of93%. Inaddition, we detected16S rDNA from the inserted DNA sequence, and identified it was alsoclosely related to Acinetobacter calcoaceticus PHEA-2with a similarity of100%. Throughchecking of16S rRNA gene, the method was prove to be reliable. This is an indirect evidencethat Acinetobacter calcoaceticus has denitrification from nitrite.