| At present,the increasing demand for high-quality protein sources of human beings has contributed to the vigorous development of the aquaculture and feed industries.Meanwhile,many practical problems of aquaculture have become increasingly prominent.Among them,excessive fat deposition in aquaculture fish is a common nutritional disorder.It not only severely restricts the healthy development of the aquaculture industry,but also potentially threat the safety of aquatic products.Therefore,understanding the principles of fat deposition in fish and carrying out studies on its regulation is a hot area in fish nutrition research.Using zebrafish as the model,this dissertation attempts to explore the mechanism of how the endocrine Ghrelin regulates zebrafish fat deposition from the perspective of endocrine and metabolic regulation,to provide a basis for an in-depth understanding of the fat deposition mechanism in fish.In this study,Ghrelin-/-knockout zebrafish modified by TALENs gene editing technique were used as experimental animals(From the Institute of Hydrobiology,Chinese Academy of Sciences).The effects of Ghrelin on lipid metabolism in zebrafish were investigated by the means of nutrition,biochemistry,molecular biology,and transcriptomics.The first step when we got the mutation zebrafish is breeding in order to develop enough fish in different life stages for the experiment.The subsequent work is divided into the following areas:1.Determining the phenotypes of larval Ghrelin-/-zebrafish.Details are: monitoring their movement behaviors,feeding with fluorescent dye-labeled paramecium,and in situ hybridization experiments for the expression of growth hormone.2.Observing the phenotypes of adult Ghrelin-/-zebrafish with normal feed.Details are: select 10 Ghrelin-/-zebrafish and normal fish(WT)of 10 dpf and the same size,feed in the same tank for 8 weeks,and measure related indicators.3.Observing the phenotypes of adult Ghrelin-/-zebrafish with High Fat Diet(HFD).Details are: separately feed the two kinds of fishes for 8 weeks,quantitatively feed(4% of body weight)with diet of different fat addition levels(7%,13%).The four treatment groups were: Ghrelin-/-high-fat group,Ghrelin-/-normal fat group,WT high-fat group,WT normal-fat group,and each group was set up with three parallel treatments.Weigh them once a week,and measure relevant indicators at the end of this feeding experiment.Finally,transcriptomic analysis were conducted on the zebrafish liver RNA extracted from the HFD group.Larval fish experimental results include the following sections:1.Parasites stained with fluorescent dye ASP were fed to the two genotypes zebrafishes.Within 30 minutes,the area and intensity of fluorescence in the digestive tract of Ghrelin-deficient zebrafish were significantly higher,indicating that their food intake was higher than that of the control group.2.The behavioral test results show that Ghrelin-deficient zebrafish were significantly more active during the daytime than the control group,but there was no significant difference at night.3.In situ hybridization experiments,the growth hormone(GH)content in the pituitary of two zebrafish larvals was similar,with no significant difference,indicating that Ghrelin was the only endogenous ligand for growth hormone secretagogue(GHSR),and its absence does not affects the expression of GH in larvals.This part of experiment shows that ghrelin participates at least two physiological functions in larval zebrafish: maintaining normal feeding behavior and neuroregulatory movement behavior.Experiment results of adult fish include three parts:1)In the mix feeding experiment,the zebrafish growth indicators of the two genotypes were not significantly different,including survival rate,average body weight,HSI(Hepatosomatic index),and VSI(viscerasomatic index).Western-blot(WB)results showed that there was no significant difference in the expression of growth hormone in the pituitary of the two genotypes;crude protein and fat of whole fish showed that no significant difference in the two genotypes of zebrafish;the fast movement distance and oxygen consumption of Ghrelin mutant zebrafish were significantly higher than those of the control group.Under the same environment,the similar growth phenotype in the two genotypes of zebrafish,suggests that there are some compensatory mechanisms to make up the loss of ghrelin function after Ghrelin gene knockout.2)There was no significant difference in growth index including survival rate,feed coefficient ratio,and VSI under feeding of different fat addition levels(7%,13%).While in the same feed group,the feed coefficient ratio of Ghrelin deficient zebrafish was less than that of the control group,demonstrating that its feed conversion rate may be higher.Tests suggest that in the HFD group,the WGR(weight gain rate)of Ghrelin-/-zebrafish was higher than that of the NFD group,and there was no significant difference among other groups.The HSI results showed that the wild-type zebrafish in the HFD group was significantly higher than the wild-type zebrafish in the NFD group,and there was no significant difference among the other groups.While respiratory rate is significantly different between every two treatments.In the NFD group,the respiratory rate of Ghrelin mutant zebrafish was significantly higher than that of the wild type group,and also in the HFD group.The respiratory rate of the wild type zebrafish in the HFD group was higher than that of the NFD group.While the respiration rate of the HFD mutant zebrafish was lower than that of the normal lipid group.It is speculated that the normal zebrafish could deal with the intake of high-energy nutrients by increasing the respiration rate,while the Ghrelin-/-zebrafish would have a metabolic disorder,an imbalance of energy homeostasis,and a decrease in respiratory rate when dealing with high-fat diets.More energy is stored in the body.The determination of its body composition showed that in the HFD group,the total protein of the whole fish of Ghrelin-/-zebrafish was significantly lower than that of the control group,and subsequent transcriptome data indeed showed that the overall protein metabolism level of Ghrelin-/-zebrafish is high;the data of the total lipid of the whole fish showed that the total fat of the Ghrelin-/-zebrafish in the HFD group was significantly higher than in the other groups,while there was no difference away the other three groups.The detection of TG content in liver and muscle showed that in the liver tissue,the TG content of the Ghrelin-/-zebrafish in the HFD group was significantly higher than that in the other groups,while there was no significant difference among the other three groups;in muscle tissue,the TG content of the Ghrelin-/-zebrafish in the NFD group was significantly higher than that in the control group,as was the case in the HFD group.The red oil section of liver tissue showed that the lipid content of Ghrelin-/-zebrafish was significantly higher than that of wild-type zebrafish in both feed treatment groups.The above results indicate that Ghrelin-/-zebrafish are more likely to deposit fat in the body when fed with high-fat diet,and have enhanced the use of proteins.3)The zebrafish liver of the high-fat diet-treated group was extracted RNA and subjected to transcriptome sequencing.Sequencing results showed significant differences in the relevant metabolic pathways between the two treatment groups.Among the differential metabolically related secondary pathways,lipid metabolism related pathways accounted for 19.995%,sugar metabolism related pathways accounted for 19.995%,and protein metabolism related pathways accounted for 60.01%.To visualize the expression of each of the important genes in the two treatment groups,a related pathway map was drawn.Compared with the WT Zebrafish,after treatment with high-fat diet,the key genes of the fat synthesis in Ghrelin-/-zebrafish are significantly up-regulated,such as FAS,DGAT1 b,etc.,while the key genes associated with lipolysis are significantly down regulated,such as CPT1,ACSL,ACADS,ACADL,energy metabolism and glycolysis,gluconeogenesis pathways are blocked,the level of the protein metabolism was significantly up-regulated,represented by the upward adjustment of the overall level of mTOR.In summary,the results of this study show that Ghrelin plays an important role to maintaining the normal feeding and normal behavior of juvenile zebrafish;the Ghrelin-/-zebrafish has normal phenotypes fed by normal feed,indicating that the mutant zebrafish has a certain compensatory mechanism and can maintain its basic metabolic level.However,under the feeding of high-fat diet,Ghrelin-/-zebrafish have metabolic abnormalities and cannot adjust the metabolic pattern in the same time as wild-type zebrafish to cope with high-energy intake,which means Ghrelin does have certain regulatory functions on zebrafish lipid metabolism.In particular,it is necessary to deal with metabolic adaptation when faced with high fat diet. |
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