Construction of cDNA library from liver of Nile tilapia Oreochromis niloticus

Hepatic cDNA library of GIFT strain Nile tilapia,Oreochromis niloticus,was constructed by Stratagene cDNA library construction kits.First,total RNA of liver was extracted by TRIZOL reagent and determined by agarose eletrophoresis and UV meter.The results showed that the purity and integrity of total RNA were both good and then mRNA was isolated with a column of oligo(dT)cellulose.Second,single-strand cDNA and double-strand cDNA were synthesized from 5' mRNA using Stratagene ZAP Express cDNA Synthesis Kit,then ligating to Eco[WTBZ]R 1 adapters,phosphorylating the EcoR 1 Ends,digesting with Xho1,size fractionating with Sepharose CL-2B gel filtration medium.Sample fractions were collected and 300 ng cDNA was obtained sufficiently to construct the library.Finally ds cDNA was ligated into Uni-ZAP XR vector and packaged into phage by Gigapack Ⅲ Gold Packaging Extract.The titer of the obtained hepatic cDNA library of Nile tilapia was 1.25×10~7 pfu/mL which agreed to the preservation and screening of the library.20 positive clones were randomly selected to carry out polymerase chain reaction and the percentage of recombinant of the library was 100%.The amplified segments were between(0.5) and 2 kb and averaged at 1.05 kb.It is indicated that the quality of the cDNA library constructed in the present study was high 80 μL amplified cDNA library was taken to excision in vitro and contained about 3.6×10~6 recombinants.The quantity of phagemid after excision was 2.0×10~6 and the efficiency of excision is 79.4%.About 1.2 mg plasmid DNA was obtained after plasmid extraction.The hepatic cDNA library provides basis of EST sequencing and cloning of metabolic enzymes and some growth factors,and then gives the insights into nutritional effects on enzyme gene expressions of the subsequent researches.