Effects Of HSL Gene On Fat Deposition In Common Carp And Its Regulatory Mechanism

Fat content is one of the important indexes to evaluate fish quality.Intramuscular fat deposition can improve muscle tenderness and succulence,which is conducive to the improvement of meat quality and taste.However,a large amount of abdominal adipose will cause the metabolic disorder and decrease in immunity and stress tolerance of fish.Moreover,it will lead to the decline of growth performance and the decrease of feed utilization,thus reducing the breeding efficiency.Therefore,it is of enormous practical significance to understand the molecular mechanism of fat deposition,and provided a theoretical basis for the improvement of fish quality and production efficiency.Fat deposition is a complex physiological and biochemical process which regulated by metabolic enzymes and genes involved in signaling pathways such as fat uptake,synthesis and decomposition.We should correct understand the fat metabolism mechanism of the key genes and signaling pathways in the process of fat deposition which is the basis for us to regulate fat deposition and improve meat quality.Hormone-sensitive lipase?HSL or LIPE?is the key enzyme and rate-limiting enzyme in the initial mobilization of fat metabolism,it is also one of the key enzymes that affect animal fat deposition.HSL decomposes triglycerides into free fatty acids and plays an important role in regulation of intracellular triglyceride content.This study took common carp as the research object,to explore HSL's spatial-temporal expression characteristics and its influence on fat deposition in common carp.Furthermore,gene knockout technique was used to move forward a single step to study the effect of HSL gene on fat metabolism and study its possible biological function and mechanism in the regulation of fat metabolism.We aimed to lay a foundation for the further study on the molecular regulation mechanism of fat deposition and the quality improvement of common carp.The main results are as follows:?1?In this study,rapid amplification of c DNA ends?RACE?technology was used to obtain full-length c DNA sequences of HSL1 and HSL2 subtypes of HSL gene,respectively.The total length of HSL1 gene c DNA is 3379 bp,including 230 bp 5'-UTR,1067 bp 3'-UTR,and an open reading frame encoding 693 amino acids.The length of HSL2 gene c DNA is 2732 bp,including 52 bp 5'-UTR,136 bp 3'-UTR and an open reading frame encoding 847 amino acids.The similarity of amino acid sequence between HSL1 and zebrafish,Japanese flounder and salmon was 88.17%,64.94%and 64.1%,respectively.The similarity of amino acid sequence between HSL2and zebrafish,Japanese flounder and salmon was 90%,73.1%and 72.3%,respectively.The amino acid sequence similarity between HSL1 and HSL2 was 60.6%.?2?Real-time fluorescent quantitative PCR was used to detect the expression pattern HSL1 and HSL2 gene in different tissues and different development period.Results showed that the HSL1 and HSL2 widely expressed in all testing tissues,but there were obvious differences.The HSL1 gene expression levels from high to low were:abdominal fat,abdominal muscles,intestine,brain,dorsal muscle,heart,spleen,liver,kidney and blood.There was no significant difference between the relative expression level of HSL1 gene in abdominal fat and that of abdominal muscle,intestine and brain?P>0.05?.The HSL2 gene expression levels from high to low were:abdominal fat,abdominal muscles,brain,heart,dorsal muscles,kidney,spleen,blood,intestine,liver.The relative expression level of HSL2 gene in abdominal fat was significantly different from other tissues?P<0.01?.RT-q PCR results showed that the common carp HSL1and HSL2 genes had two different expression patterns at the early developmental stages.The expression levels of HSL1 gene m RNA showed a downward trend,among which the 8-cell period was the highest,with a significant difference compared with other periods?P<0.05?,and the lowest at the feeding stage.Different from HSL1,the m RNA expression levels of HSL2 gene in the early developmental stage of common carp showed a trend of decrease and then increase.The expression of HSL2 m RNA was the highest at 8-cell period and the lowest at 7-segment stage.After that,it returns to a higher level during the feeding stage.It is speculated that HSL1 and HSL2 genes have different expression patterns in common carp.?3?We investigated the relationship between HSL and fatty deposition in common carp.In the abdominal adipose tissue,the expression level of HSL1 m RNA in the low-fat group was significantly higher than that in the high-fat group?P<0.01?.The m RNA level of HSL2 in the low-fat group was significantly lower than that in the high-fat group?P<0.01?.In the dorsal muscle tissue,there was no significant difference in HSL1 m RNA level between the high-fat group and the low-fat group?P>0.05?,while the HSL2 m RNA level in the high-fat group was significantly higher than that in the low-fat group?P<0.01?.We suggested that HSL1 and HSL2 genes have different mechanisms in the regulation of abdominal fat deposition in common carp.?4?Taking zebrafish as the model,we explored the effect of HSL2 gene knockout on fat deposition.The mutant pure line of zebrafish HSL2 gene?-80/+4 bp?was constructed by using CRISPR/Cas9 gene editing technology.Morphological results showed that the homozygous mutant HSL2^-/-zebrafish did not show up abnormal embryonic development and early growth.There was no significant difference in body length and body weight between 2 months old HSL2^-/-and wild type?P>0.05?.Muscle fat metabolism indicators showed that,compared with the wild type,the total fat content was increased?P>0.05?.The content of triglyceride?TG?decreased and the difference was significant?P<0.05?,while the content of diglyceride?DAG?increased but the difference was not significant?P>0.05?.In addition,the content of HSL was decreased significantly?P<0.01?.?5?We investigated the effect of HSL2 gene mutation on the expression of upstream and downstream genes in HSL phosphorylation pathway in zebrafish.In muscle tissue,compared with the wild type,HSL2^-/-zebrafish HSL2 gene m RNA expression was significantly reduced?P<0.01?.The m RNA expression levels of PKA,PKG and ATGL genes in the upstream were extremely significant decrease?P<0.01?;m RNA expression levels of downstream MGL and a FABP genes were both extremely significant decrease?P<0.01?.The m RNA expression level of AQP7 gene increased,but the difference was not significant?P>0.05?.Preliminary results showed that the knockout of HSL2 gene blocked the lipidolysis pathway,which resulted in the decrease of the expression level of the downstream genes,inhibited the hydrolysis of DAG,and thus increased the content of DAG.The results will provide reference for the study of the mechanism of HSL gene in the fat deposition of common carp.