Establishment And Application Of Intestinal Cell Lines And Gill Cell Lines Of Plectropomus Leopardus

The leopard coral grouper(Plectropomus leopardus),one of few farmed marine speices,is a precious kind of warm water reef fish mainly distributed from the Persian Gulf and the Pacific Ocean.Nevertheless,with progressive breakthroughs of reproduction technology and enlarging breeding scale,emerging diseases break out in breeding and producing,which seriously hampers the sustainable development of seabass breeding industry.The fish cell line is a in vitro culture system with lowcost and high repeatability.It has high application value in virology,immunology,oncology,genomics and toxicology owing to its precise manipulation capability.In this study,using cell line techniques,we establish intestinal and gill cell lines,and preliminary study were made to determine optimal growth conditions.Moreover,sensitivity to different kinds of bacteria and heavy metals was subsequently analysed.Main research contents and results show as follows:1.In this study,The leopard coral grouper intestinal cell lines(PLI)and gill cell lines(PLG)were established by tissue block digestion and adhesion methods.Morphological observation showed that both PLI and PLG were rendered as fibrous,passing over 40 generations.For PLI and PLG,the optimum serum concentration was 20%,the optimum culture medium was L15 and the optimal growth temperature was 26℃.2.The 18 S rRNA gene fragments of the two cell lines were compared with the18 S r RNA gene sequences of P.leopard in NCBI database,and the homology was100%,which validated their origin from P.leopard.Karyotype analysis exhibited chromosome aneuploidy in both cell lines,indicating deletion,breakage and distortion,while still maintained a characteristic number of 48 chromosomes.By analyzing the cell cycle of PLI and PLG,the results showed that both PLI and PLG cell lines were composed of proliferative diploid cells,which could be used for continuous culture and genetic studies.3.The enhanced Green Fluorescent protein(EGFP)reporter gene was successfully transfected into two cell lines by liposome transfection method.Transfection efficiency of PLI cells and PLG cells was about 20% and 15%respectively.Bacterial sensitivity analysis showed that PLI cells and PLG cells were sensitive to Vibrio harveyi and Edwardii tarda,with higher sensitivity to Vibrio harveyi.Heavy metal susceptible analysis showed that PLI and PLG were susceptible to all three heavy metals(Cd,Cu and Hg),and there are more susceptible to Hg.And there exited a dose-dependent manner between the cell viability and the concentration of these three heavy metals.In summary,this study triumphantly established a leopard gill spines and gill perch intestinal cell lines.Both two cell lines could be utilised as ideal in vitro research tools to launch subsequent researches in gene function,toxicology,immunology and other associate disciplines,thereby proffering original experimental materials and approaches to other fish cell line establishment.