| China is the largest country of pig farming and pork consumption.With the improvement of the pork quality requirements of consumers,the production of high-quality pork has become an important issue that the pig industry urgently needs to solve.Intramuscular fat is an important index for evaluating the quality of pork,and it is closely related to meat quality such as tenderness and flavor.Genetic improvement of intramuscular fat trait has been the focus of attention in pig genetics and breeding.However,the genetic improvement of porcine intramuscular fat traits using conventional breeding techniques has been unsatisfactory for a long time.Therefore,identification of key genes that affect the intramuscular fat traits and the development of valuable molecular markers have a very important significance for the early and accurate breeding of intramuscular fat traits,the accurate genetic improvement of intramuscular fat traits and for the improvement of pork quality.In this study,variant sites and key candidate genes affecting intramuscular fat of pigs were identified using whole-genome resequencing and transcriptome sequencing technology.The results lay the theoretical foundation for further development of the molecular regulation mechanism of intramuscular fat formation and molecular marker technology.1.Identification of key genetic variation sites affecting intramuscular fat in pigs using genome-wide resequencingIn this study,1,310 PDLY pigs were used as study population and their intramuscular fat from the longissimus dorsi muscle were extracted using Soxhlet extraction.Thirty individuals with extremely high or low intramuscular fat were screened from 1,310 individuals and intramuscular fat content was extremely significant between them(P<0.01).Two libraries were constructed after equal pooling of 30 individuals in the high and low groups,respectively,and was sequenced on Hiseq 2000.Sequencing results indicated that high(H)and low(L)groups yielded 849,292,378 and 861,810,316 Clean Reads,respectively.By comparison with pig reference genome,75.56%and 79.56%of Clean Reads in the two libraries were mapped onto the reference genome,respectively.In addition,the coverage of the reference genomes was 89.11%and the sequencing depths were 26.12x and 25.66x in H and L groups,respectively.Single nucleotide polymorphisms(SNPs)analysis showed that 2,038,236 and 2,021,681 SNPs were obtained in the high and low groups.Among SNPs,74%SNPs were located in the intergenic region in the two groups,while 94%SNPs in the gene were located in the intron.242,010 and 240,975 Indels were further identified in the high and low groups,respectively,and the distribution ratio on the genome was similar to that of the SNPs.The average density of SNPs per chromosome was similar in the H and L groups,while it was different in the different chromosomes;the average density of SNPs on chromosome X was the lowest.The average density of Indels per chromosome was similar in the high and low groups,but there was a large difference in the Y chromosome.A total of 17,184 candidate SNPs affecting intramuscular fat were identified by comparison between the H and L groups,and 1,149 genes were annotated.The GO analysis of these genes can be annotated into three categories including biological process(BP),cellular component(CC),and molecular function(MF),and were further classified into 49 subcategories.Among them,cell and cell terms include the most number of genes.The GO enrichment analysis showed that 137,18,and 31 GO terms were enriched in the BP,CC,and MF.The result of KEGG enrichment of these genes indicated that 26 pathways were significantly enriched,including PPAR signaling pathways,etc.2.Identification of candidate genes affecting intramuscular fat of pig using transcriptome sequencing technologyIn this study,279 PDLY pigs were used as study population and housed in 11 groups,and their intramuscular fat from the longissimus dorsi muscle were extracted using Soxhlet extraction.The results of statistical analysis showed that the maximum,minimum and average intramuscular fat content were 6.56%,1.09%,and 2.64%,respectively.In addition,the standard deviation(SD)and coefficient of variation(CV)value of intramuscular fat content were 0.91 and 34.55%,respectively.In this study,we first randomly selected 3 groups from 11 groups,and selected 3 extremely high and low intramuscular fat individuals(named H and L)and then they were mixed into a pool for subsequent sequencing library.A total of 6 sequencing libraries were constructed.Phenotypic statistics showed that intramuscular fat content was significantly different between them(P<0.01).The sequencing results showed that a total of 268,826,758 Clean Reads were obtained from the six sequencing libraries(the number of clean reads per sample was 44,222,498-45,247,560),a total of 40.33 G clean bases;in addition,Clean Reads accounted for more than 90%in Raw Reads.By further comparison with pig reference genome,the average match rate of all samples was approximately 65%.A total of 13,568 novel transcripts and 16,798 expressed genes were detected in the six sequencing libraries in this study.Differentially expressed genes(DEGs)analysis results indicated that 75 DEGs were identified between the H and L groups,including 28 upregulated and 47 downregulated.The results of GO enrichment analysis of DGEs showed that 27,27,and 39 GO terms were significantly enriched in BP,CC,and MF.Among them,FASN is an important candidate gene that affects intramuscular fat,which is enriched in acyl-[acyl-carrier-protein]hydrolase activity and fatty acid synthase activity.KEGG enrichment analysis of DGEs revealed that 13 pathways were significantly enriched,of which the most significant pathways were Insulin signaling pathway,PPAR signaling pathway,etc.These pathways included SCD,PL1N1,PDK1,and PRKAR2A.3.Study of the effect of candidate gene PLIN1 on intramuscular fat content in pigsA total of 30 individuals with extremely high or low IMF were selected from the 279 PDLY pigs and intramuscular fat content was extremely significant between them.PLIN1 mRNA levels in the high and low IMF groups were detected using qRT-PCR technique and results indicated that the mRNA level of PLIN1 was significantly higher in individuals with high(H)IMF content than in individuals with low(L)IMF content(n=30).The PLIN1 protein levels were further examined in the H and L IMF groups and the results showed that its protein level was significantly high in the H IMF group compared with the L IMF group.PLIN1 is extremely highly expressed in adipose tissue of pigs and lowly expressed in liver tissue,but hardly expressed in other tissues.In addition,PLIN1 mRNA and protein expression levels were significantly increased after induction of differentiated adipocytes compared with undifferentiated intramuscular preadipocytes.The localization of PLIN1 in the longissimus dorsi muscle was detected using immunohistochemistry,and results indicated that PLIN1 is localized in adipocyte between muscle fibers in the longissimus dorsi muscle and is not localized in the muscle.The localization of PLIN1 protein in intramuscular adipocytes was detected using immunofluorescence and the results showed that PLIN1 protein is localized around the lipid droplets in the intramuscular adipocytes.The PLIN1 gene was knocked down in intramuscular adipocytes using siRNA technology and results showed that the mRNA and protein levels of PLIN1 were significantly lower in the siPLINl group than in the control group.When PLIN1 was knocked down,the triglyceride(TG)levels in the intramuscular adipocytes were significantly reduced compared to the control group.After PLIN1 knockdown,lipid droplets became smaller and the number of small lipid droplets were increased.Moreover,there were no significant expression change in the GPAT1,DGAT1,ATGL,and HSL genes which were involved in triglyceride metabolism,and only mRNA and protein levels of FASN were significantly decreased.4.Study of the effect of candidate gene FASN on intramuscular fat content in pigsFASN protein expression levels in the H and L IMF groups were detected by Western blot and results indicated that its expression level in the H IMF group was significantly higher than that in the L IMF group(n=3).FASN mRNA levels in 60 individuals(including 30 H IMF and 30 L IMF individuals)were detected and found that FASN mRNA level was higher in the H IMF group compared with the L IMF group.In addition,the FASN gene is highly expressed in adipose tissue,and its expression level was increased after intramuscular adipocyte differentiation.The FASN mRNA and protein expression levels were significantly down-regulated using siRNA knock-down technology.The proliferation and cycle of intramuscular adipocytes were detected using EdU staining and flow cytometry after inhibiting FASN expression and the results showed that the proliferation of intramuscular adipocytes was decreased,and its cell cycle also significantly blocked.In addition,knockdown FASN can down-regulate p-AKT protein expression levels,while AKT protein levels did not change significantly.p-AKT protein expression was significantly upregulated in intramuscular adipocytes treated with SC79,an activator of the AKT pathway.Activation of the AKT pathway can rescue the proliferation inhibition and cycle arrest of intramuscular adipocytes caused by FASN knockdown. |
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