| Feed costs account for about 70% of the total in modern swine production.Improving feed efficiency and reducing feed costs is an important method to promote pig feed efficiency.Skeletal muscle is the largest tissue in the body,and its energy consumption has a percentage of about 60% of total energy metabolism.Reducing energy metabolism of pig skeletal muscle is a significant way to improve feed efficiency.MiRNAs(miRNAs)are small conserved RNA molecules of 20-24 nucleotides,which can play important regulatory roles in animals and plants by targeting m RNAs for cleavage or translational repression.Lactate dehydrogenase(LDH),low-density lipoprotein(LDL),triglyceride(TG),creatinine(CRE),glucose(GLU)are widely involved in glucose metabolism and fat metabolism,and is closely related to energy metabolism.Our previous results showed that the expression of miR-208 b is increased in high feed efficiency in swine skeletal muscle,whereas the majority of the mitochondrial genes are down-regulated,and the activity of the lactate dehydrogenase enzyme are significant difference in pig skeletal muscle between the individuals of the significant different feed conversion efficiency.In this study,we used the GWAS technology,combined with the analysis of gene function in cellular and individual levels to reveal the molecular mechanism of miR-208 b,LDH,LDL,TG,CRE and GLU in the regulation of energy metabolism in skeletal muscle,and to provide an important theory for pig feed efficiency traits.The results in this study are as follows:(1)In adult Large Whites,we did tissue expression profiling of miR-208 b in heart,liver,spleen,lung,kidney,stomach,intestine,lymph,gastrocnemius muscle,dorsal muscle,extensor digitorumlongus,soleus muscle,fat and other tissues by Northern blot,the results showed that miR-208 b expressed in heart,gastrocnemius,longissimus muscle,extensor digitorumlongus,soleus muscle and other tissues,and the heart have the highest expression level,however,the expression of miR-208 was negligible in other tissues.(2)We predicted the target genes of miR-208 b by Target Scan software.In the BHK-21 cells,MED13 was verified as the target gene of miR-208 b by dual luciferase reporter gene assay.MED13 is associated with the thyroid hormone pathway,suggesting that miR-208 b may be related to thyroid hormone signaling pathway.(3)20 Babl/c mice were randomly divided into four groups,5 mice each group: the first and second test groups were given 150,600 ug T3 / kg BW intraperitoneal injection,T3 was dissolved in 0.17 m M Na OH;and the third group(NC)were administered by intraperitoneal injection of 0.17 m M Na OH as negative control;the fourth group(Mock)were treated as a blank control.These experiments lasted two weeks.The results showed that NC group and Mock groups had no significant differences in FCR;the feed efficiency of 150 ug T3 / kg BW group were significantly lower than that in NC group,with a decrease of approximately 21.3%;the feed efficiency of 600 ug T3 / kg BW group were greatly significantly lower than that of NC group,falling by about 38.1%,indicating a certain concentration of thyroid hormones can reduce feed conversion efficiency.(4)40 SD rats were randomly divided into five groups,8 rats each group: the first,second,third and fourth groups were given 300,30,3,0.3mg MMI / L of water treatments,respectively,the fifth group received normal drinking water(0mg / L)as a control,these experiments lasted 28 days.The results showed that,when the MMI concentration was0.3mg / L,the feed conversion rate significantly increased by about 9.4% than that of the control group,which illustrated that,feed efficiency could be improved after a certain concentration of MMI inhibited thyroid hormone.(5)RNA was extracted from the leg muscles of SD rats in 0.3mg MMI / L of water treatment group and control group.Afterwards,RNA was reverse transcribed to c DNA to detect changes in the expression level of miR-208 b,β-MHC,α-MHC,UCP-2,PGC1-α by quantitative PCR several.The results showed that the expression level of miR-208 b andβ-MHC in 0.3mg MMI / L water treatment group was significantly upregulated;while the expression level of PGC1-α,UCP-2 and α-MHC was significantly downregulated than the control group.These indicated that the thyroid hormone signaling pathway was involved in the formation of fast and slow muscle fiber,and initially speculated that the downstream genes which regulates energy metabolism of thyroid hormone signaling pathway might be UCP2 and PGC1-α.(6)We measured 5 serum biochemical indicators(LDL,GLU,TG,CRE,LDH)related with energy metabolism of a population with 187 Large Whites,and applied correlation analysis with RFI and FCR trait indexes,the results showed that blood biochemical indicators significantly associated with RFI were GLU,LDL and TG;and blood biochemical indicators significantly associated with FCR was GLU.(7)We used GAPIT to treat serum biochemical indicators LDL,GLU,TG and LDH by GWAS analysis in a population of 187 Large White.Three to five SNP loci and the nearest genes with the most significant association with related indicators were selected,and we found 4 genes(LRRN3,GATA3,ADRA1 D and CNTNAP5)may be associated with serum biochemical indicators candidate genes.(8)In other research work during my graduate study,I compared 7 different methods on primer design for RT-q PCR from successful rate of detection,linear dynamic range to sensitivity.The results demonstrated that method F(Stem-loop S-poly(A)-tail RT-PCR)and method B(Liner S-poly(A)-tail RT-PCR)are generally better,but they both had different advantages: if the miRNA content is lower,method B is better because of its higher sensitivity;and if it’s difficult to design primers due to the structure of miRNAs,method F is better as it has a wider range of adaptation and higher success rate.This study reveals the relationship between miR-208 b and feed efficiency and thus provides a theoretical basis for the future work.We find that LRRN3,GATA3,ADRA1 D and CNTNAP5,as the preliminary candidate genes of RFI and FCR,and hence,it provides a certain reference value for the screening of candidate genes. |
