Copmarative Proteomic Profiles Of The Gill In Grass Carp(ctenopharyngodon Idella)response To Hypoxic Stress And The Research On Tetraploid Induction Of Grass Carp

Grass carp(Ctenopharyngodon idella)belongs to Cypriniformes,Cyprinidae,Ctenopharyngodon,which has the advantages of the rapid growth of succulent and tender meat,intermuscular thorn less advantages,as one of the main types of freshwater fish farming in Chinese.In the actual breeding process,fish are often challenged to survive in variable environments,with fluctuating dissolved oxygen(DO)concentrations,which is very easy to cause the fish to die of hypoxia.At present,less attention has been given to the levels of the molecular mechanisms of acute hypoxia adaptation in fishes.Gills as the main respiratory organs of fish,participate in all kinds of important physiological activities,are capable of extensive remodeling in response to changes in DO.After hypoxic stress,the levels of proteins expression and signaling pathway in gill can change.Polyploid breeding technology belongs to the research field of Genome engineering.Tetraploid grass carp can not only self-breed,but also hybridize with diploid grass carp,quickly and efficiently obtain triploid grass carp and triploid grass carp.There are obvious advantages in terms of growth rate,resistance and population yield,which has created a new type of fish genetic breeding method.The study includes following three contents mainly:(1)In order to establish and optimize the two-dimensional electrophoresis(2-DE)system for proteomic analysis on the gill of Grass carp,protein samples of grass carp gill were separated by lysis.Immobilized pH gradient(IPG)strips was used to perform isoelectric focusing electrophoresis(the first dimension),and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)served as the protein separation.By optimizing different lysate formulations and protein purification methods,different immobilized pH gradients(IPG)gel strips,as well as varied loading amount and staining methods were investigated.The proteins were successfully isolated from grass carp gill and were separated by 2-DE.Image Master 2D Platinum 7.0 analysis software was applied to analyze the 2-DE images.The results showed that the quality of 2-DE profiles was significantly improved by replacing Tris and TBP with DTT in lysis buffer,loading 150μg samples on the pH4-7 immobilized pH gradients(IPG)strips,and staining with silver nitrite.In consequence,compared with other methods the 2-DE gels profiles with wider pH molecular weight range,lower background noise,higher resolution and more applicable for variant analysis were obtained by using this method consequently.(2)In order to find the key role of protein information in grass carp gill response to hypoxic condition,and to explore the possible molecular mechanism,two-dimensional gel electrophoresis(2-DE)combined with liquid chromatography-mass spectrometry(LC-MS/MS)technique was used to identify the differential proteomics of grass carp gill after hypoxic stress.Protein spots obtained from hypoxia-stressed group were 372±11,and control group 406±14 by means of lmageMaster2 D Platinum7.0 analysis.The expressions of 15 spots expressed differentially of hypoxia-stressed group varied significantly after challenge.In addition,these differential proteins were identified by mass spectrometry,then searched in database.The result demonstrated that the expression of Toll-like receptor 4,Ephx1 protein,Isocitrate dehydrogenase,L-lactate dehydrogenase,GTP-binding nuclear protein Ran and Glyceraldehyde-3-phosphate dehydrogenase were up-regulated remarkably,however,Keratin type II cytoskeletal 8,Type I cytokeratin,ARP3 actin-related protein 3 homolog,Thyroid hormone receptor alpha-A,ATP synthase subunit beta,Citrate synthase,Tropomyosin 2 and Tropomyosin 3 were down-regulated remarkably.Six proteins are found in the hypoxia inducible factor-1(HIF-1)signaling pathway.It was concluded that grass carp gill is involved in the process of energy generation,metabolic process,cellular structure,anti-oxidation,immunity,signal transduction and other processes in response to hypoxic stress.This is the first time to report on proteomics analysis of expressed proteins in the gill of grass carp,and this study will help to understand the molecular mechanisms of hypoxic stress responses at protein level in fish.(3)In order to confirm the feasibility of heat shock induced large-scale production of grass carp tetraploids,based on the heat shock conditions that were found in the laboratory to induce a relatively high survival rate of hatching grass carp embryos,the fertilized eggs were treated to inhibit the first time Mitosis,the specific method is as follows: incubation temperature 21±1°C,embryo fertilization 40 min,42min,51 min,respectively,using 41 °C and 42 °C heat shock treatment 2min.Hatched fry were cultured in big ponds.In winter,the ploidy of grass carp induced by heat shock was analyzed and analyzed for relative DNA content.The results showed that only diploid and triploid grass carp were obtained by heat shock induction,and tetraploid grass carp was not obtained.The relative DNA content of triploid grass carp was about 1.5 times that of normal diploid grass carp DNA.In this study,heat shock treatment was used to induce the treatment of grass carp fertilized eggs,and a certain proportion of triploid grass carp was obtained,but the overall proportion was not high,and tetraploid grass carp could not be obtained so far.It can be seen that the heat shock method is practically difficult for the production of tetraploid grass carp.Follow-up research on grass carp polyploid breeding can explore new options and try other polyploid breeding methods.