Screening And Identification Of MiRNAs Associated With Intramuscular Fat Deposition In Chickens

Objective: As a key substance that affects muscle flavor,intramuscular fat(IMF)has a significant effect on meat quality and is an important indicator for evaluating the quality of meat products.However,in the early stage of chicken embryo development,the expression of miRNAs and their target genes related to IMF deposition is still less.In this study,the leg muscles of Rose-crowned chicken and Cobb chicken in early embryonic development was used as the object.Key miRNAs and their target genes related to IMF deposition were obtained by using high-throughput sequencing technology,and they involved the pathways and biological processes were also be researched.It is expected to explore the molecular mechanism of intramuscular fat(IMF)regulation of muscle flavor,and use miRNAs to target intervention of IMF sediment-related genes to provide new ideas for improving muscle flavor research.Methods:(1)The content of intramuscular fat,inosinic acid and amino acid,reducing sugar and thiamine were respectively determined by Soxhlet extraction,high performance liquid chromatography and kit method.(2)The time point when the two kinds of chicken leg muscles and pectoralis muscles fat droplets begin to deposit was determined by histological observation of two kinds of chicken muscles flaps.(3)Two small RNA libraries for chicken leg muscle were constructed,and the sequencing results were analyzed by bioinformatics analysis method,and the reliability of the library was verified by RT-PCR.(4)The target gene of gga-miR-3532-5p was predicted by using miRanda,PITA and RNAhybrid software,and the target gene was identified by the dual-luciferase reporter system.Results:(1)The total amino acids,essential amino acids and umami amino acids of Rose-crowned chicken were all higher than that of Cobb chicken(P<0.05),the content of IMP and IMPc was also higher than that of it.In the content of thiamine and reducing sugar in Rose-crowned chicken was higher or equal to that in Cobb chicken,the content of intramuscular fat(IMF)is also mostly higher than that in it.In general,the quality of Rose-crowned chicken is superior to that of Cobb chicken.The five flavors of the two hens were mostly higher than those of the roosters,and the contents of IMF of two kinds of chicken muscles partially increased with the day age.(2)Both types of chicken leg muscles began to have fat droplet deposition on the 7th day of embryo development,while pectoralis muscles began to the 8th day.Besides,lipid droplet deposition in muscles showed a "wave-like" trend with the increase of embryo age.The deposition rate of Rose-crowned chicken was higher than that of Cobb chicken,but the deposition rate was lower than that of it.(3)Small RNA sequencing results showed that: the MGT vs KGT library obtained 306 miRNAs with significant difference in expression(154 up-regulation and 152 down-regulation),including 273 known miRNAs,33 new predicted miRNAs;the MMT vs KMT library,335 miRNAs with significant differences in expression were obtained(171 were up-regulated and 164 were down-regulated),including 286 known miRNAs,and 49 newly predicted miRNAs.The GO and KEGG pathways showed that the differentially expressed miRNAs were mainly involved in embryo development and IMF deposition pathways such as PPAR signaling pathway.The results of qRT-PCR verification of six miRNAs with significant differential expression were consistent with the results of high-throughput sequencing.(4)gga-miR-3532-5p and its target gene FABP5 related to IMF deposition were screened.The dual-luciferase reporter gene identification showed that the reported gene activity of FABP5-3 '-UTR region was significantly inhibited by gga-miR-3532-5p,and resulting in a 19.2 % downregulation of luciferase activity(P<0.01).It indicates that gga-miR-3532-5p does have a targeted relationship with FABP5 gene and inhibits its expression.Conclusion:(1)the nutritional value and muscle flavor of Rose-crowned chicken were better than that of Cobb chicken,and the hen of two kinds of chicken is better than the cock.(2)the leg muscles began to have lipid droplet deposition on the 7th day of embryonic development,and the pectoral muscles began to the 8th day,and the 7th day of embryonic development can be a key point in the study of lipid droplet deposition.(3)The small RNA libraries of the two chicken leg muscles were successfully constructed,and miRNAs with significant difference in expression were obtained,including 306 in MGT vs KGT libraries and 335 MMT vs KMT in libraries.(4)It is proved that FABP5 is the target gene of gga-miR-3532-5p,and gga-miR-3532-5p may participate in the negative regulation of IMF deposition by acting on FABP5.