| Highly unsaturated fatty acids play an important role in the growth,development and reproduction of Huanghe carp(Cyprinus carpio).In particular,the content of highly unsaturated fatty acids such as EPA,DHA and ARA are an important index representing the quality of Huanghe carp.The growth character of fish is also one of the most concerned characters in aquaculture.Cultivating economic aquatic varieties with fast weight gain can greatly improve aquaculture profits and economic benefits.At present,genes related to the synthesis and metabolism of highly unsaturated fatty acids in other fish such as Huanghe carp have been screened,and the structural characteristics,expression level and cell function characterization of genes related to highly unsaturated fatty acids traits have been preliminarily explored in other species such as model organism zebrafish.However,there are few studies on the function verification of genes related to highly unsaturated fatty acids traits at the cell level in Huanghe carp,and the specific mechanism is not clear.Therefore,this study takes the Huanghe carp individuals with significant differences in EPA and DHA contents as the research object.By integrating the whole genome association analysis and transcriptome sequencing data before the research group,the differentially expressed genes related to EPA and DHA traits are screened,and then the relative expression of candidate genes in liver,brain and muscle of Huanghe carp with significant differences in EPA and DHA content was detected by real-time fluorescence quantitative experiment.Then,by predicting the target mi RNA of elovl5-a and lpl-a gene3’UTR sequence of Huanghe carp,combined with the verification results in relevant literature,the mi RNA with high prediction score and high reliability was selected for the double luciferase experiment of elovl5-a and lpl-a gene.At the same time,using CRISPR/cas9 gene editing technology to construct gene mutants of Huanghe carp with deletion of elovl5-a,fabp1 b.1,acat1,lpl-a and acsbg2 genes.The DNA of brain,liver and muscle tissues in the experimental group and the control group were extracted,and the individuals with successful gene editing were determined by PCR amplification and Sanger sequencing.Muscle tissues of mutant and wild individuals were taken for fatty acid detection,and the difference of fatty acid content was analyzed;then the liver RNA of mutant and wild individuals was extracted,and the relative expression of mutant gene was detected quantitatively by real-time fluorescence.This study analyzed the pathway and gene interaction mechanism of highly unsaturated fatty acids synthesis such as EPA and DHA in Huanghe carp.The results provide a theoretical basis for the breeding of Huanghe carp varieties with high content of EPA and DHA.This study also analyzed the transcriptome data of brain,liver and muscle tissue of two groups of Huanghe carp with significant weight difference were analyzed by RNA-seq technology,and the differentially expressed genes were mined by bioinformatics analysis method.The relative expression of candidate genes related to growth traits of Huanghe carp was identified by real-time fluorescence quantitative experiment.The results can provide data support for the breeding of Huanghe carp.The main results of the study are as follows:(1)The acsbg2 gene was screened to be related to EPA traits of Huanghe carp.15 genes were associated with DHA traits of Huanghe carp,including angptl4,acsbg2,fabp1 b.1,acat1,elovl5-a,elovl5-b,lpl-a,lpl-b,slc27a1,fads6-a,fads6-b,elovl6,fabp3,rxrgb,elovl2.And elovl5-a,elovl5-b,lpl-a and lpl-b refer to elovl5 and lpl genes located on different chromosomes.elovl2,elovl5-a,elovl5-b and elovl6 belong to the fatty acid lengthening enzyme gene family,and lpl-a and lpl-b belong to the lipase gene family,fads6-a and fads6-b belong to desaturase gene family;fabp1b.1,fabp3 belongs to the fatty acid binding protein gene family,and angptl4 belongs to the angiopoietin like gene family,and acsbg2,acat1,slc27a1 and rxrgb genes play a role in the process of DHA fatty acid synthesis.(2)The fluorescence quantitative results of candidate genes showed that the expression of acsbg2 corresponding to EPA character in the liver tissue of Huanghe carp was significantly higher in the group with higher fatty acid content than that in the group with lower fatty acid content(P < 0.05);the expression levels of elovl6 and fabp3 in the group with high DHA content were significantly higher than those in the group with low DHA content(P < 0.01);the expression of rxrgb in the group with high DHA content was significantly higher than that in the group with low DHA content(P < 0.001).However,the expression of acat1 and fabp1 b.1 in the group with high DHA content was significantly lower than that in the group with low DHA content(P < 0.01);there was no significant difference in the expression of elovl5-a,fads6,slc27a1,acsbg2 and lpl-a genes between the two groups(P > 0.05).In the muscle tissue of Huanghe carp with significant difference in DHA content,the expression of angptl4 in the group with high DHA content was significantly higher than that in the group with low DHA content(P < 0.01).However,the expression of slc27a1 in the group with high DHA content was significantly lower than that in the group with low DHA content(P < 0.01);there was no significant difference in the expression of acsbg2 gene between the two groups(P > 0.05).Compared with the brain tissue of Huanghe carp with higher DHA content,the expression of elovl5-b gene in the brain tissue of Huanghe carp with lower DHA content increased significantly(P < 0.05).There was no significant difference in the expression of fads6,elovl2 and lpl-b genes between the two groups(P > 0.05).In conclusion,acsbg2,elovl6,fabp3,rxrgb,angptl4,elovl5-b and other genes play an important role in the synthesis and metabolism of EPA and DHA high unsaturated fatty acids in Huanghe carp.(3)The results of double luciferase gene report experiment of 293 T cells showed that mir-26a-5p could negatively regulate elovl5-a gene and mir-27b-3p could negatively regulate lpl-a gene.It shows that the way of elovl5-a and lpl-a genes involved in the regulation of highly unsaturated fatty acids content of Huanghe carp is also affected by mi RNA.(4)Finally,elovl5-a and fabp1 b.1 were successfully constructed by CRISPR / cas9 gene editing technology.The number of mutants Huanghe carp with gene deletion is six.Compared with wild-type population,fatty acid test results show that C18:3n3(α-linolenic acid),C20:4n6(arachidonic acid,ARA,AA),C20:5n3(eicosapentaenoic acid),EPA and MUFA(including C16:1,C18:1n9c,C20:1,C22:1n9 and C24:1)had no significant difference in two groups.However,the content of C22:6n3(docosahexaenoic acid,DHA)in the mutant population was higher than that in the wild-type population.The results of real-time fluorescence quantification showed that the expression of elovl5-a and fabp1 b.1 in the liver of the mutant population of Huanghe carp was higher than that of wild population.However,the expression of acat1 gene in mutant individuals was lower than that in wild populations.The reason why the m RNA expression of elovl5-a and fabp1 b.1 gene may be that its fluorescent quantitative primers detect the expression of other copies of the gene at the same time.According to the results,it is speculated that the reason for the increase of DHA content in the mutant population of Huanghe carp may be except elovl5-a and fabp1 b.1 gene are involved in the pathway of DHA synthesis in Huanghe carp,and there has other lengthening enzymes and desaturating enzymes make effects.The accumulation of DHA high unsaturated fatty acids in the gene editing group led to the down-regulation of acat1 gene expression.The results showed that elovl5-a,fabp1 b.1,acat1 gene plays an important role in regulating the content of EPA and DHA in Huanghe carp.The down-regulated expression of acat1 gene in the mutant group was helpful to the accumulation of DHA fatty acids in the body.elovl5-a,fabp1 b.1,acat1 gene may play an important role in regulating the content of EPA and DHA in Huanghe carp.(5)Through bioinformatics analysis and real-time fluorescence quantitative verification of go and KEGG transcriptome data of growth traits of Huanghe carp,13 candidate genes and their corresponding signal pathways such as MAPK,Wnt and erb B are closely related to the growth traits of Huanghe carp,namely mapk1,fgf13,mapk7,wnt8 b,ednra,tgfbr2,mapk10,wnt10 b,wnt16,mapk12,notch2,fgf19 and lpl. |
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