| In the current intensive farming model, fish often appear many problems such as lipid metabolism disorder of liver. Grass carp as the second fish production in the world. Breeding widely used intensive mode of compound feed, although grass carp nutritional needs and formulation technology research has been carried out widely, but still often appear in production related to feed problem of sugar and lipid metabolism disorder and liver lipid metabolic disorder, and have affected the quality of grass carp. In this study, we use grass carp liver by high concentration carbohydrate and lipid diet induced as the experimental materials. By high-throughput sequencing technology, obtained specific miRNA, which are related to lipid metabolism. Using prediction software to predict the target gene of miRNA, screening and identification specific miRNA, which regulating SREBP-1 mediated lipid metabolism in fish. At the same time combined with biochemical tests and its enzyme activity determination methods, detection of lipid metabolism-related genes, such as PPARγ SREBP-1, ACC1, FAS, LPL and CPT-1, screening out lipid metabolism-related miRNAs, which we have to do the analysis of function verification and regulation mechanism, in order to interpret miRNAs regulate lipid metabolism function and molecular mechanism in grass carp, fill the lipid metabolism blank of miRNAs in fish. This study from the selection of key miRNA of regulating lipid metabolism, to study the effect of exogenous nutrients affect fish fat utilization and its mechanism, the research data can enrich the carbohydrates and lipid metabolism regulation mechanism of fish, and is expected to provide a theoretical basis for improving the utilization efficiency of carbohydrates and lipid metabolism in fish feed, for preventing and treating liver steatosis and other nutrition metabolic disease provided a theoretical basis, and provide a scientific basis for healthy growth in fish.1. High concentrations of carbohydrates and lipid induce liver steatosis in grass carp(Ctenopharyngodon idella)To explore concentrations of carbohydrates and lipid feed on grass carp induce liver fat accumulation and metabolic regulation mechanism. This study designed four purified test diets were formulated to contain proximate 32% of protein levels, but have different levels of carbohydrates and lipid, Control(31% carbohydrate, 4% crude lipid), H-LIP(31% carbohydrate, 8% crude lipid), H-CHO(45% carbohydrate, 4% crude lipid), H-CL(45% carbohydrate, 8% crude lipid). The feeding trial was conducted for 65 days. After the feeding experiments, detecting changes of grass carp growth, feed efficiency ratio(FER), protein efficiency ratio(PER) and liver fat accumulation, preliminary judgment whether the formation of fatty liver on the grass carp, the results showed that grass carp growth performance, FER, PER HSI,VSI were not significantly increased(P > 0.05), but the H-CL diet group had the highest MFI among the all groups(P < 0.05), and there is a significant increase in liver fat than the control group(P < 0.05). So high concentrations of carbohydrates and lipid induce can lead to liver fat accumulation in grass carp.We used the automatic biochemical analyzer(AU-640, OLYMPUS) determine the serum concentrations of triglyceride(TG), globulin(GLB), total protein(TP) and the activity of alanine aminotransferase(ALT), aspartate aminotransferase(AST). The results showed that AST/ALT, GLB, TG, TP et al abnormalities. Description liver has suffered serious damage, with H-CL group has the most serious damage.Using paraffin embedded and HE staining and observing in liver histology under the microscope, the result showed that the treatment group compared with the control group of lipid droplets in the liver was significantly increased and hepatocytes severe swelling deformation. Indicated that high concentrations of carbohydrates and lipid induce liver steatosis in grass carp, and may also cause the liver function injury.2. By high-throughput sequencing technology, detection the influence of miRNA expression for using high concentrations of carbohydrates and lipid feed feeding grass carpAfter the feeding experiment,extract and separate the RNA of the grass carp liver, bulid the small RNA library, using the Hiseq sequencing technique, screen out the differential expression miRNA with high concentrations of carbohydrates and lipid induced. We screen out 531, 455 and 637 differential expression miRNAs compared with control, and 18 novel miRNAs.3. Detection the miRNA and lipid metabolism-related genes of liver in grass crap with realtime fluorescence quantitative PCRDetection the expression of six kinds lipid metabolism-related genes and three kinds mi RNA with RT-PCR, the result show that the expresion of lipiogenic factors(SREBP-1, PPARγ) and lipiogenic enzymes(ACC1, FAS) is up-regulation, and the expression of lipolysis enzyme(LPL) is up-regulation too, but the expression of lipxoygenase enzyme(CPT-1) is down-regulation, the result demonstrated that high concentrations of carbohydrates and lipid can cause lipid metabolism related genes expression of the response, so as to regulate carbohydrates and lipid metabolism of grass carp. Three kinds miRNA which are differential expression in whole transcriptome resequencing was verified, miR-122, miR-370 and miR-33, the result reveal that the expression of three kinds mi RNA significantly increased with high concentrations of carbohydrates and lipid induced, this result is identical to the whole transcriptome resequencing, the result show that miRNAs and lipid metabolism related genes may participate in the carbohydrates and lipid metabolism process of grass carp.4. Molecular cloning of SREBP-1 gene and predicting the action point of miRNA in its 3 ’UTR region of grass carpSterol regulatory element binding proteins(SREBPs) are nuclear transcription factors critical for regulating the expression of genes related to glucose and lipid metabolism. In order to investigate the molecular regulatory mechanisms of lipid metabolism and liver lipid accumulation in fish, the cDNA sequence of sterol regulatory element binding protein 1(SREBP-1) from grass carp has been identified by homology cloning and rapid amplification cDNA ends(RACE), and the structural features of the gene and its encoded protein have been analysed through bioinformatics methods. Using the method of real-time PCR, the expression pattern of SREBP-1 in eight different tissues of grass carp has been studied, and predicted the action point of miRNA in its 3 ’UTR region of grass carp. The result revealed that the cDNA of SREBP-1 in grass carp is 4760 bp in length(In GenBank accession number is KJ162572), which consisted of a 3426 bp open reading frame(ORF), encoding 1141 amino acids. The amino acid sequence has a typical basic helix-loop-helix leucine zipper(bHLH-zip). Alignment based on amino acid sequence showed that grass carp SREBP-1 had a homology of 76%-88% to its counterparts of other fish. Phylogenetic analysis showed that grass carp SREBP-1 clustered with that of zebrafish. Real-time PCR result showed that the SREBP-1 mRNA could be detected in all the examined tissues of grass carp. But the expression level in brain was highest, followed by liver and intestines, and with little expression in the kidney, spleen, muscle, fat and gonad tissues. There are two kinds of lipid metabolism-related mi RNAs exist at the 3 ’UTR region of SREBP-1, miR-33 and miR-16.5. SREBP-1 gene 3’UTR dual luciferase report vector constructionThis study successfully constructed the SREBP-1 3’UTR luciferase reporter gene vector, and SREBP-1 3’UTR region sequence contains binding sites of miR-33, verified SREBP-1 is the candidate target genes of miR-33, can at the cellular level to validate the function of miR-33 at the next step. |
PDF Full Text Request