| Sheep is an important breed of livestock.It can provide us with a variety of products such as meat,milk,skin and wool.For meat-type sheep,growth and reproduction are the main goals of breeding and selection.However,genetic factors associated with the sheep growth and lean meat productions have not been fully identified.Therefore,studying metabolic pathways and genes that regulate growth and meat production will provide new strategies for improving the yield and quality of mutton.Genes on the leptin-melanocortin signaling pathway are critical in weight regulation and energy homeostasis.Relevant genes in this pathway,especially melanocortin 4 receptor(MC4R),signal transducer and activator of transcription 3(STAT3),and leptin(LEP)genes can regulate food intake by affecting hypothalamus activity and energy expenditure.Based on relevant reports in humans and other species,it is necessary to identify mutations in genes related to the leptin-melanocortin signaling pathway and the effects of these mutations on body measurement and fat thickness traits in sheep.In particular,to clarify the location,structure,function,as well as expression of these genes in various tissues of the sheep,and to identify those mutations in the promoter region of the MC4 R gene that have a significant effect on the transcriptional regulation of this gene.In this experiment,a total of 551 sheep from 5 different China meat-type sheep breeds were involved from their native habitats.The variations in these genes were investigated using a Polymerase Chain Reaction(PCR)direct sequencing method.General Linear Models(GLM)was used to examine associations between variation in these genes and differences in various body measurement and fat deposition traits.To detect the relative m RNA expression of MC4 R and STAT3 genes,we collected samples from seven tissues(hypothalamus,kidney,liver,heart,lung,semimembranosus,and longissimus muscles)from birth to six months age(n = 42).Apart from,to confirm the STAT3 protein localization and expression in different tissues,we employed immunohistochemistry(IHC)technique.Furthermore,to delineate the minimal proximal MC4 R promoter region,five plasmids were constructed using PGL-3 basic vector,transfected to cultured HEK293,and C2C12 cells.The minimal MC4 R regulatory region was identified based on the plasmid fragment activities in the dual luciferase assay.Overall,the following results have been found in this study.(1)In the first study(Chapter 2),14 genes(LEP,LEPR,TUB,NPY,Ag RP,POMC,JAK,SH2B1,STAT3,MC4 R,MC3R,MRAP2,SIM1,and BDNF)involved in the leptin-melanocortin signaling pathway has been screened for possible association with growth and muscular high-yield carcass traits in sheep.The functional genomics analysis of these candidate genes was done using different bioinformatics tools.Each gene was examined for potential polymorphisms based on re-sequence data(n=71)of different sheep populations.The LOSITAN was used to detect the loci under selection.The gene ontology(GO)analysis indicated that these candidate genes were associated with complex biological processes,such as regulation of feeding behavior,energy homeostasis,and positive regulation of the c AMP biosynthesis.These distinct biological processes are well known for regulation of body weight,growth,and fat deposition traits,which clearly explained the mechanism underlying rapid weight gain and muscular highyield carcass in sheep.Besides,a total of 7,226 SNPs were detected in these 14 genes.Using the fixation index(Fst)based method,MC4 R,STAT3,SH2B1,and TUB genes were found to be an outlier and under significant positive selection with the provisional combined allocation of mean heterozygosity and Fst.This study provides a novel perspective for a better understanding of the biological processes linked with rapid weight gain and muscular high-yield carcass traits in sheep.This knowledge is conducive to the improvement of yield and sheep meat composition.(2)In the second study(Chapter 3),the exon,3‘untranslated(3‘UTR),and promoter region of the ovine MC4 R gene were screened for genetic variation in 480 sheep from five breeds.A total of 14 SNPs,including five coding variants(SNP1: g.9C >T,SNP2: g.12 C >G,SNP3: g.93 G >A,SNP4: g.519 C >G,SNP5: g.682 G >C),and one SNP(SNP6: g.1016 G >A)in the 3‘UTR,and 8 SNPs(-1131C>T;-1038G>A;-1036G>T;-1026G>A;-943G>T;-287G>A;-206G>A,and-103C>G)in the promoter(2000bp in the 5‘ flanking)region of the gene were detected.The genotype-phenotype association analysis results demonstrated that in the missense variation at g.519 C >G locus,the GG genotype animals have significantly higher body weight(BW),withers height(WH),heart girth(HG),body length(BL),rump length(RL),and rump width(RW)(p < 0.01)than that of AA genotype animals for such parameters.Similarly,there was a significant variation between the GG and AA genotypes for backfat thickness(BFT)(p < 0.05).At the g.682 G >C locus,animals with genotype GG possess significantly greater rump height(HR),and backfat thickness than those with CC genotype(p < 0.05).Besides,the promoter activity assay result showed that the minimal proximal promoter region was from-1207/-880 bp upstream of the first exon.The real-time quantitative PCR(RT-q PCR)data shown that the MC4 R relative m RNA had differentially expressed between sex and age.(3)In study 3(Chapter 4),genetic variations in STAT3 has investigated and verified their contribution to sheep body measurement and fat deposition traits.We have detected 24 SNPs both in the intron and exon region of this gene.The degree of conservation of STAT3 was estimated,followed by measurements of its m RNA expression using the quantitative Real-Time Polymerase Chain Reaction(q RTPCR)and Immunohistochemistry.Both the q RT-PCR and IHC results consistently evidenced the ubiquitous expression of the STAT3 in all tested tissues,and the highest expression was recorded in the liver.Moreover,the STAT3 was differentially expressed across ages and sex.In this study,four novel nonsynonymous single nucleotide polymorphisms(ns SNPs)were detected.The association analysis results also demonstrate that the missense mutations in STAT3 had a statistically significant effect on the sheep body weight and body size measurement traits(p<0.05).This is the first report that exhibits associations between STAT3 genetic markers and body weight as well as fat-related traits in sheep.(4)In study 4(Chapter 5),the genetic variations at the LEP gene were evaluated for associations with growth and fat deposition traits in sheep.In this experiment,11 nonsynonymous single nucleotides(ns SNPs)were detected from the re-sequence data of 71 sheep individuals.These detected ns SNPs have screened at a total of 250 individuals from 5 different sheep breeds.The association analysis results show that the majority of the ns SNPs were significantly associated with one or more growth and fatness traits in the sheep.Notably,One SNP(CT at base pair 257)leads to higher levels of growth traits while the TT base pair is associated with the elevated fat score.Besides,in the in silico analysis,3 ns SNPs(g.257C/T,? T86 M,g.292G/A,? D98 N,and g.470C/A,? P157Q)were resulted in increasing protein stability.These findings,coupled with the association analysis,suggesting that these ns SNPs may increase the evolvability of the gene by allowing the protein to accept a broader range of beneficial mutations while still folding to its innate structure.Taken altogether,in this study,we tried to better describe the genetic basis of sheep growth and lean meat production.Our data suggested that the leptin-melanocortin signaling pathway genes,notably,these three genes(MC4R,STAT3,and LEP)described in-depth in this thesis could be used as gene-markers in sheep breeding to select for desirable body weight and fat-related traits.However,as the regulation of growth and muscular high-yield carcass traits are multifaceted.Many additional molecules and neural circuits are involved,and it will be increasingly important to determine the role of these complex systems in the sheep physiology.More importantly,making use of the identified genes in sheep breeding program through marker-assisted and genomic selection should receive due attention. |
PDF Full Text Request