| Seed industry is the core of aquaculture development.Their innovation is essential for the healthy development of aquaculture.Rapid growth,high disease resistance and good meat quality has always been the pursuit of aquatic breeders.Aquatic biotechnologies,especially the development of gene editing technology provides us with a new idea of variety improvement.Compared with traditional breeding method,gene editing techniques can instantly get the varieties exhibit good characters byadjusting the phenotype of aquatic animals directionally at the genetic level.However,in the field of aquatic breeding,gene editing technology is still in its infancy,large amount of functional genes need to study and testify by this technology.In this thesis,we chose zebrafish as experimental model for their transparency,short reproductive cycle,perfect genetics databases and constructed the mutant zebrafish models of the genes related to growth and development of fat and muscle using CRISPR/Cas9 technology.On the one hand,this study aims to screen some target genes that can be used to improve varieties by comparing and analyzing differences in growth traits between wild type and mutant zebrafish.The advantage of these mutant models,we can further excavate and improve the function of the genes and its related regulation pathways,which will provide databases for gene function annotation.The main results were showed as follow:1.We successfully deleted the genes of mstna,mstnb,mc4r,mc3r,mrap2a and mrap2b in zebrafish using CRISPR/Cas9 technology,and generated the heritable and homozygous depletion lines by hybridization screen.2.Deletion of mstnb but not mstna enhanced growth performance,which were performed on somewhat higher and wider body trunk.The histological examinations by sectioning and H&E staining showed that the numbers of the muscle fibers increased dramatically with smaller size in the mstnb-/-zebrafish.The removal of mstnb significantly reduced the resting metabolic rate in zebrafish.3.The expunction of mstnb caused a high accumulation of lipids and a reduction of muscle glycogen in the muscle tissue,as well as a raise of the concentration of glucose in the blood.However,deletion of mstna found an opposite result.The expression of mstna inmstnb-/-zebrafish and mstnb inmstna-/-zebrafish were increased,which indicated an existed a negative influence between mstna and mstnb.4.Although survival rates under normal conditions were slightly decreased in both mstna-/-and mstnb-/-zebrafish,mortality after dexamethasone-induced stress was increased by?30%.Furthermore,transcriptional levels of several critical immune-related genes were decreased,and the ability to withstand exposure to pathogenic E.tarda was decreased,compared with that of controls.In mstnb-/-but not mstna-/-zebrafish,expression of NF-?B subunits and several pro-inflammatory cytokines failed to respond to E.tarda exposure except nfkb1,c-rel and tnf?.5.Deletion of mc4r enhanced growth performance under overfeeding conditions but not normal feeding conditions.Furthermore,the food intake in both male and female mc4r-/-zebrafish were increased by 56.8%and 27.6%,respectively.The transcriptional levels of several anorexigenic genes?pomca,leptinaand leptinb?were decreased under both fed and fasting conditions and an orexigenic gene?npy?was still activated even under fed conditions.Additionally,the oxygen consumption rate was increased in mc4r-/-zebrafish and the percentage of weight loss of mc4r-/-fish after 15 day's starvation was increased by21.47%.6.Deletion of mc4r significantly increased the number of eggs that one female fish produced,but no differences were observed in the fertilization rate and hatching rate.The gonad index of female mc4r-/-zebrafish was also increased.The histopathological analysis of sections of ovaries and testes in mc4r-/-fish showed that the proportion of each stage follicles did not change,but the number of follicles were increased in the same magnified visual field.7.Deletion of mrap2a effected the expression of genes related to HPA axis,which could be rescued by microinjection of mrap2a mRNA.After exposure a vortex stimulus,the activation of crh,cyp17,cyp11a1 and hsd3b were inhibited in mrap2a-/-zebrafish,and the secretion of cortisol was also suppressed.8.Deletion of mrap2a or mrap2b all could decrease the protein expression levels of mc4r.Overexpression of mrap2a could increase the expression levels of mc4r,as well as overexpression of mc4r could also increase the transcriptional expression levels of mrap2a.The expression of crh could be enhanced in wild type zebrafish by overexpression of mc4r or mrap2a,but not in mc4r-/-zebrafish by overexpression of mrap2a.However,co-microinjection of mc4r and mrap2a could increase the expression of crh in mc4r-/-zebrafish.All above results suggested that only in the presence of mc4r can mrap2a regulate the expression of crh.Although deletion of mstnb weakened the immunity of zebrafish,it could increase the body size and the fat content in the muscle,reduce the oxygen consumption rate,which is necessary for improving varieties.Thus,we considered mstnb as a candidate gene for improving traits in economic fish.Additionally,only when providing sufficient food,could deletion of mc4r increase growth rate of zebrafish,which will increase the production cost in the actual production.In this study,we did not observe any obvious growth advantage in mrap2a-/-and mrap2b-/-zebrafish,on contorary,we found that mrap2a can take part in fish stress reaction by regulating the function of mc2r and can affect the function of mc4r with mrap2b.We also found that mrap2a and mc4r are involved in the expression of crh,then associated with multiple endocrine axis.This study not only screened an important candidate gene used for improving varieties,but also provided the baseline and reference for the application of the CRISPR/Cas9 techniques in fish breeding,as well as provided theoretical help for revealing the regulation process between mrap2a,mrap2b and mc4r,and their disturbing effects on the endocrine system. |
PDF Full Text Request