To explore the association between STAT3 single nucleotide polymorphism and lactation traits of sheep,414 lactating sheep were selected as test subjects,the expression of STAT3 in seven tissues including mammary gland was detected by RT-qPCR,the single nucleotide polymorphism of STAT3 was detected by sanger sequencing,STAT3 was typed by Penta-primer amplification refractory mutation system PARMS,and the effect of the nucleotide sequence variation of the gene on milk performance traits was analysed.RT-qPCR results revealed that STAT3 expression was highest in lungs,followed by liver and mammary tissues,and lowest in spleen.A total of five novel SNPs,c.22+537 C/T,c.22+658 C/T,c.318+126 C/G,c.417+360 G/A and c.417+395 T/C,were identified in the sheep STAT3,and three genotypes were detected at each SNP locus.Association analysis revealed that ewes with the genotype CC at the c.318+126 C/G locus had a higher milk fat rate than those with GG,and CC-type at the c.22+537 C/T locus,CC-type at the c.22+658 C/T locus,GG-type at the c.417+360 G/A locus,and TT-type at the c.417+395 T/C locus all had higher milk fat percentage and dry matter content.The haplotype combination H1H7 exhibited significant positive effects on milk fat percentage,dry matter content,and ash content.Additionally,the average daily milk yield of individuals carrying the haplotype combination H1H2 was increased by 0.266 kg compared to those carrying H1H7.In conclusion,the five nucleotide variation sites of STAT3 screened in this study may provide a theoretical basis for molecular selection of milk performance traits in milk sheep.

In order to clarify the role of PRKAA1 gene in lipid deposition in goat intramuscular precursor adipocytes,the complete CDS region of the goat PRKAA1 gene was successfully amplified and cloned using RT-PCR,followed by comprehensive bioinformatics analyses. Subsequently,PRKAA1 gene overexpression vectors were constructed utilizing a double enzyme digestion approach and transfected into intramuscular precursor adipocytes.Real-time Fluorescence Quantitative PCR(RT-qPCR)was used to measure the expression level of PRKAA1 gene in the dorsal longissimus dorsi muscle in juvenile and young adults,and the expression level of PRKAA1 gene and lipid metabolism-related genes in precursor adipocytes was also detected in the process of inducing the differentiation of adipocytes. The role of PRKAA1 gene in the formation of lipid droplets was observed and investigated using oil red O staining. The results showed that the total nucleotide sequence length of goat PRKAA1 gene was 2 520 bp. Among them,the 5' untranslated region(UTR)contained 3 bp,the coding region(CDS)was up to 1 680 bp,while the 3' untranslated region(UTR)was 837 bp,and the protein encoded by this gene consisted of 559 amino acid residues. Phylogenetic analysis of the goat PRKAA1 gene revealed that sheep was the most closely related species,followed by cow,pig,dolphin,human,mouse,and rat,whereas chicken was the most distantly related species. The PRKAA1 gene was expressed at a lower level in 9 month-age than 2 month-age in the longest dorsal muscle,and expression was present throughout the induction of differentiation of precursor adipocytes and was at its highest on day 6. In experiments overexpressing the PRKAA1 gene,the content of lipid droplets in precursor adipocytes in goat muscle was significantly reduced. In addition,the expression of genes related to lipid metabolism,such as SREBP1-c,FASN,ACC,AGPAT6,PLIN1,ATGL,and ACOX1 were significantly down-regulated,and the changes were statistically significant. However,the expression levels of several genes,DGAT1,DGAT2,LPL and HSL,did not show significant changes. These results suggest that overexpression of the PRKAA1 gene might affect lipid deposition through multiple metabolic pathways,specifically,it inhibits the ab initio synthesis of fatty acids,decreases the rate of fat synthesis,and increases the rate of fat degradation. These mechanisms work together to inhibit lipid deposition in goat intramuscular precursor adipocytes.

In order to clarify the regulatory role of marginal zone B and B1 cell specific protein(MZB1)in intramuscular adipose differentiation of goats,the study first cloned the full-lenth sequence of goat MZB1 gene and analyzed the sequence to obtain the relevant biological characteristics;then used Real-time Quantitative PCR(qPCR)to construct the expression profile of goat MZB1 in tissues and cells;finally,the goat MZB1 overexpression plasmid was transfected into the target cells,followed by differentiation induction;the effects of MZB1 overexpression on the differentiation process were evaluated through morphological analysis and qPCR.The results showed that the goat MZB1 gene sequence was 860 bp in length,including 570 bp CDS region,encoding 189 amino acids;goat MZB1 protein was not only acidic but also negatively charged,which could be stably existed in the cells.Goat MZB1 expression was detected in eight tissues,including the heart and the liver,and the highest expression was found in the spleen.The expression of MZB1 in goat intramuscular adipocytes after 72 h of oleic acid-induced differentiation was significantly higher than that before oleic acid-induced differentiation.Goat MZB1 overexpression increased the number and size of lipid droplets in goat intramuscular adipocytes;the expression levels of FASN,PPARγ,C/EBPα,SREBP1,and C/EBPβ were highly significantly upregulated.The above results suggest that MZB1 plays a positive regulatory role in goat intramuscular adipocyte differentiation,and this positive regulation may be achieved by increasing the expression of the adipose differentiation marker genes PPARγ,C/EBPα,C/EBPβ,and the fatty acid synthesis marker genes SREBP1 and FASN.

Muscle formation depends on the interplay of various cellular and extracellular signals and factors.This study investigates the regulatory relationship between Myocyte enhancer factor 2A(MEF2A)expression levels and promoter methylation at both tissue and cellular levels,to provide theoretical references for the genetic development of Guanling cattle.Guanling cattle were used as experimental subjects.Initially,the expression levels of the MEF2A in Guanling cattle tissues were analyzed in conjunction with their promoter methylation status.Subsequently,overexpression and interference vectors of the MEF2A gene were successfully transfected into primary myoblasts of Guanling cattle,and the effects of MEF2A expression level changes on promoter methylation levels were analyzed.The results showed that the MEF2A expression levels in various tissues of young Guanling cattle were higher than those in adult cattle,while the methylation rate of MEF2A in young cattle tissues was lower than that in adult cattle,with DNMT1 expression trends consistent with this.Additionally,increased MEF2A expression led to a decrease in its methylation rate,whereas decreased MEF2A expression resulted in an increase in its methylation rate.This study confirms from both tissue and cellular perspectives that the expression level of the MEF2A gene in Guanling cattle is negatively correlated with its methylation rate,providing a theoretical basis for genetic marker-assisted genetic improvement of cattle.

To elucidate the role of the MYL2 gene in muscle development and high-altitude adaptation in yak,and to explore its potential impact on meat quality and production performance,this study conducted cloning and tissue expression analysis of the MYL2 gene in Pamir yak.By cloning its coding sequence(CDS)and performing bioinformatics-based functional prediction and tissue-specific expression profiling,the functional characteristics of the MYL2 gene were further clarified.Additionally,it seeks to analyze and explore the structure and function of the MYL2 gene.Real-time quantitative PCR(RT-qPCR)technology was employed to quantify the expression levels of the MYL2 gene in various tissues of the Pamir yak,including the heart,liver,spleen,lung,kidney,pancreas,and longissimus dorsi muscle.The results indicated that the coding sequence(CDS)length of the Pamir yak MYL2 gene was 501 bp,which encoded a protein of 166 amino acids.Comparative homology analysis revealed that the MYL2 gene of Pamir yak shared the closest genetic relationship with that of the wild yak,exhibiting a similarity of 100%,while the most distant genetic relationship was observed with the chicken.The protein analysis results indicated that the molecular formula of the protein was C₈₄₂H₁₃₁₃N₂₁₉O₂₆₁S₇,with a molecular mass of 18.904 42 ku and a theoretical isoelectric point of 4.831.The instability index was measured at 28.3,suggesting that the protein was stable.Additionally,the protein contained one N-glycan modification potential site and 15 potential phosphorylation sites.Furthermore,its structure did not exhibit a transmembrane domain or signal peptide,and it was primarily localized in the cytoplasmic membrane and cell wall.The secondary structure was predominantly composed of 93 α-helices(56.02%),60 random coils(7.83%),and 13 β-sheets(36.14%).RT-qPCR results showed that MYL2 gene expression was extremely significantly higher in the heart and longissimus dorsi muscle of Pamir yak compared to other tissues,suggesting that this gene may play a key regulatory role in maintaining cardiac function and promoting skeletal muscle growth.Based on protein function prediction,MYL2 is likely involved in the development of cardiac and skeletal muscles by regulating the expression of muscle structural proteins,thereby contributing to the maintenance of locomotor capacity and thermogenic performance under cold,hypoxic plateau conditions.

In order to analyze the alterations in the expression profile of circular RNA(circRNA)in chicken liver cancer cells line(LMH)infected by fowl adenovirus serotype 4(FAdV-4),and the regulatory role of circRNA in the FAdV-4 infecting process,transcriptome sequencing was carried out on FAdV-4-infected LMH cells and uninfected ones.Enrichment analysis of GO functions and KEGG signaling pathways was executed for differentially expressed circRNAs,and five randomly selected circRNAs were verified by Real-time Fluorescent Quantitative PCR(qRT-PCR).The results demonstrated that the circRNAs in the infected and uninfected groups were distributed on the preponderance of chromosomes,and their lengths were mainly concentrated between 300 and 1 000 bp.Differential expression analysis identified 72 circRNAs,with 32 showing significantly upregulated expression levels and 40 presenting downregulated expression levels.GO functional analysis revealed that the genes from which the differential circRNAs originated were mainly enriched in processes such as cellular processes,metabolic processes,catalytic activity,and nucleic acid-binding transcription factor activity.KEGG pathway analysis indicated that the differentially expressed circRNAs were primarily enriched in the Notch signaling pathway,RNA degradation,and the MAPK signaling pathway.The qRT-PCR results showed that the expression levels of the five verified circRNAs were consistent with the sequencing results,further validating the reliability of the sequencing results.This study analyzed the expression profile of circRNAs in FAdV-4-infected LMH cells and screened out differentially expressed circRNAs,providing data support for exploring the functions of circRNAs during the FAdV-4 infection process and the interaction mechanism between the host and FAdV-4.

The aim is to establish a rapid and accurate method for detecting Avian hepatitis E virus(aHEV)antibodies.The ORF2 protein of the CaHEV-GDSZ01 strain was expressed in an E.coli prokaryotic expression system and utilized as the coating antigen.The reaction conditions were optimized to determine the optimal working conditions,leading to the establishment of an indirect ELISA method for detecting aHEV chicken serum antibodies.The specificity,sensitivity and repeatability of the method were tested,and compared with Western Blot method,chicken clinical serum samples were preliminarily detected.The results showed that ORF2 protein was successfully expressed and purified,and Western Blot assay confirmed that ORF2 protein could specifically react with aHEV-positive chicken serum.The optimal reaction conditions for ELISA were as follows:ORF2 protein was coated with 200 ng per well at 4 ℃ overnight(12 h);blocked with 5% nonfat milk at 37 ℃ for 1-2 h;serum was diluted at 1∶1 600,incubated at room temperature for 1 h;rabbit anti-chicken IgY-HRP was diluted at 1∶5 000,incubated at 37 ℃ for 90 min;TMB was incubated at 37 ℃ for 20 min.The ELISA method could detect the positive sera of aHEV diluted 51 200 times,and did not cross-react with positive sera of other chicken-borne viruses such as NDV,AIV-H5,AIV-H9,ALV,REV,and MDV.The intra-batch and inter-batch coefficients of variation were both less than 10%,rate between the Western Blot method was 95.12%. Clinical serum samples from chicken farms in different areas of Guangdong were tested by this ELISA method, and the total positive rate reached 66.34%.In conclusion,this study successfully established a simple,effective,specific,sensitive and reproducible serological detection method for aHEV infection,which can provide technical support for the surveillance,prevention and control of aHEV infection at grassroots.

Fat storage inducing transmembrane protein 2 (FITM2) plays an important role in regulating lipid storage and skeletal muscle energy metabolism.To scan the polymorphisms of the FITM2 gene in sheep and investigate its relationship with growth traits in Hu sheep,the experiment selected 1 128 healthy and disease-free,genetically well-documented Hu sheep as the experimental group,and used the FITM2 gene in sheep as the research object.First,the expression differences of the FITM2 gene in different tissues were analyzed using qPCR technology.Then,the genetic polymorphisms of the FITM2 gene in sheep were detected using PCR amplification,Sanger sequencing,and AQP (allele-specific quantitative PCR-based genotyping assay).Finally,the association between the genetic polymorphisms and growth traits was analyzed.The research results showed that the FITM2 gene was expressed in different tissues of Hu sheep,with the highest expression level in the tail fat and significantly higher than that in other tissues.The gene polymorphism detection results showed that there was a C>T mutation at position 72704027 in the first intron of the FITM2 gene.The results of correlation analysis showed that the 100,120 d body weight,140,160 d body height,80,120,140,160 d body length,and 100 d chest circumference of CC genotype individuals were significantly higher than those of TT genotype individuals.In summary,the FITM2 gene g.72704027 C>T mutation site in sheep can be used as a candidate molecular marker related to growth traits in Hu sheep,providing a theoretical basis for the genetic breeding work of Hu sheep.

To investigate the function of tachykinin 3 receptor gene(TACR3)and the expression and distribution of its encoded product neurokinin B receptor(NK3R)in the oviduct and uterus of Ganjia Tibetan sheep,hypothalamus,oviduct and uterus tissues during estrous cycle and anestrus of Ganjia Tibetan sheep were taken as samples.The TACR3 gene coding sequence(CDS)was cloned by nested PCR.The biological function of TACR3 interacting proteins was predicted using STRING protein interaction database,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Real-time Quantitative PCR(qRT-PCR),Western Blot and immunohistochemistry(IHC)were used to analyze the distribution and expression of TACR3 gene and NK3R in the oviduct and uterus during estrous cycle and anestrus of Ganjia Tibetan sheep.The results showed that the total length of the open reading frame of TACR3 gene in Ganjia Tibetan sheep was 1 395 bp,encoding 464 amino acids,and no signal peptide structure was found,with seven transmembrane structures.TACR3 may participate in biological processes such as neuropeptide signaling pathway,GnRH signaling pathway and calcium signaling pathway.The NK3R protein encoded by TACR3 was mainly expressed in the mucosal epithelium,ciliary cells,secretory cells of oviduct,uterine glands and stromal cells of uterus.In oviduct,the expression level of TACR3 mRNA was the highest in the metestrus,and the expression level of NK3R protein was the highest in the estrus.The expression levels of TACR3 mRNA and protein in uterus were the highest in the metestrus.The results showed that TACR3 was conserved in animal evolution,and TACR3 mRNA and protein were expressed in oviduct and uterus tissues at different estrus periods,which may play an important role in the reproductive process of Ganjia Tibetan sheep.

The objective of this study was to identify differentially expressed mRNAs,miRNAs,and lncRNAs between different tail types of sheep in their tail fat tissues,and to construct a critical ceRNA(competing endogenous RNAs)regulatory network involved in the regulation of fat deposition in the tails of sheep.Three 2-year-old(24-month-old)Dorper sheep and three Tianjin large-tail sheep were randomly selected.Tail fat tissues were collected for H&E staining and whole transcriptome sequencing.Bioinformatics approaches were employed to screen for differentially expressed RNAs,predict target genes,and conduct functional enrichment analyses.The regulatory network and targeting relationships were further analyzed from the perspective of lipid deposition regulation.The results demonstrated that the tail fat cell area of Tianjin large-tail sheep was significantly larger than that of Dorper sheep.There were a total of 1 156 differentially expressed mRNAs between the tail fat tissues of Dorper sheep and Tianjin large-tail sheep(with 403 upregulated and 753 downregulated),72 differentially expressed miRNAs(46 upregulated and 26 downregulated),and 392 differentially expressed lncRNAs(142 upregulated and 250 downregulated).Additionally,216 target genes were predicted from the differentially expressed lncRNAs.These target genes were involved in biological processes related to lipid metabolism,such as the tetrahydrofolate metabolic process,GAIT complex,and coenzyme catabolic process,as well as signaling pathways like glycerolipid metabolism,biotin metabolism,fat digestion and absorption,and the phosphatidylinositol signaling system.The study successfully constructed a ceRNA network closely associated with sheep tail fat deposition by predicting the targeting relationships between differentially expressed mRNAs and lncRNAs and miRNAs.Among them,24 lncRNAs and 6 miRNAs were predicted to regulate genes closely related to lipid metabolism,including PROX1,LPL,and LRP1B.In summary,PROX1,LPL,and LRP1B play a key role in the deposition of tail fat in sheep.This study predicts that four lncRNAs,including MSTRG.9916.7,positively regulate fat metabolism through PROX1,while MSTRG.7563.1 and MSTRG.4729.1 positively regulate fat metabolism through LPL.

The aim of this study was to clone the silent mating type information regulator 2-related enzyme 7(SIRT7)gene,detect its expression pattern in different tissues and investigate its effect on milk fat synthesis in mammary epithelial cells of dairy goats.The results showed that by designing the primers of SIRT7 gene,the sequence of SIRT7 gene in dairy goat was successfully cloned,with 1 376 bp and a coding region of 1 203 bp,encoding 400 amino acids and showing the highest homology with sheep.SIRT7 was a hydrophilic protein,free of signal peptides and transmembrane structures,with secondary structures such as irregular coils,α-helices,extended chains and β-turns.The relative expression of SIRT7 gene was the highest in the liver tissues of dairy goats,and it also had certain expression levels in other tissues by qRT-PCR.The pcDNA3.1-SIRT7 overexpression vector was successfully constructed and transfected into mammary epithelial cells of dairy goats.Compared with the control group,the mRNA level of SIRT7 gene in the overexpression group was up-regulated about 33 times.After SIRT7 gene overexpression,the expression of stearoyl-CoA desaturase-1(SCD1)gene was significantly decreased,and the expression levels of cluster of differentiation 36(CD36),diacylglycerol acyltransferase 1(DGAT1),sterol regulatory element binding protein 1(SREBP1)genes extremely significantly reduced.The content of triglyceride in cells was significantly decreased.The results of oil red O staining showed that the accumulation of lipid droplets around the nucleus was significantly reduced.Thus, SIRT7 gene inhibited lipid metabolism in mammary epithelial cells of dairy goats,which laid a foundation for further exploration of the regulatory mechanism of SIRT7 in mammary epithelial cells of dairy goats.

The purpose of this study was to screen the candidate genes and key pathways related to traits at first laying through transcriptomic analysis of liver tissues on laying and non-laying Kangle yellow chickens,and to provide a theoretical basis for studying the molecular mechanisms of laying traits regulation in the liver of chickens.It also provides certain reference for the selection and breeding of Kangle yellow chickens.Three individuals of liver tissues at 154 days of age that had started laying (Group H) and had not started laying (Group L) were selected.Total RNA was extracted using the TRIzol method,and transcriptome sequencing was performed using the Illumina sequencing platform.Differentially expressed genes between the two groups were identified.The differentially expressed genes were subjected to functional enrichment and protein-protein interaction analysis.Nine candidate genes were randomly selected,and their expression levels were verified by qRT-PCR in the livers of the nine individuals.A total of 21 465 genes were detected to be expressed in the liver tissues,and a total of 227 differentially expressed genes were identified,among which 48 were up-regulated and 179 were down-regulated.The qRT-PCR validation results showed that the expression trends of nine genes in the two groups of individuals were basically consistent with the RNA-Seq results,and they showed a decreasing or increasing trend in the H,M(to be laid),and L three groups.Six candidate genes for the age at egg-laying trait were initially identified,namely VTG1,VTG2,VTG3,APOV1,RBP,and RNF186.The five crucial signaling pathways were fat digestion and absorption,cholesterol metabolism,ECM-receptor interaction,estrogen signaling pathway,D-glutamine and D-glutamate metabolism.Six genes and five key signaling pathways were preliminarily identified related to the traits at the first laying of Kangle yellow chicken.

The aim of this study was to explore the reproductive adaptability of PEDV in porcine intestinal epithelial cells(IPEC-J2)and to evaluate the inhibitory effect of Polygonum cuspidatum water extract on it in vitro.The effects of different trypsin concentrations and different time on the activity of IPEC-J2 cells and the expression of PEDV N protein in cells were evaluated by cytotoxicity assay and Western Blot assay.The virus titer,PEDV N gene transcription level and N protein expression were detected by TCID₅₀,Real-time Fluorescence Quantitative PCR and Western Blot.The effect of water extract of P.cuspidatum on the proliferation activity of IPEC-J2 cells was detected by CCK-8 method.Further,the inhibitory effect of P.cuspidatum water extract on the expression of PEDV N gene and N protein was detected by Real-time Fluorescence Quantitative PCR and Western Blot experiments,and the effect of P.cuspidatum water extract on each stage of PEDV life cycle was explored.The results showed that when the concentration of trypsin was 2 μg/mL,trypsin had no effect on the cells.At this time,PEDV showed the best reproductive adaptability and reached the highest virulence value after 36 h.When the concentration of P.cuspidatum water extract was lower than 100 μg/mL,there was no cytotoxicity at 12,24 h.The water extract of P.cuspidatum significantly inhibited the expression of N gene under Co-treatment and Post-treatment,and significantly inhibited the expression of PEDV N gene mRNA and N protein in a dose-dependent manner.The water extract of P.cuspidatum had a significant inhibitory effect on PEDV N gene mainly in the replication and proliferation stage of the virus life cycle,and had no significant inhibitory effect in other stages.In summary,2 μg/mL trypsin had the best adaptability to PEDV reproduction,and the water extract of P.cuspidatum had a significant inhibitory effect on PEDV in vitro,which mainly acted on the replication and value-added stage of PEDV life cycle.

MAP3K7 is a member of the MAP3K family and plays an important role in fat differentiation,inflammation and tumorigenesis.However,its relationship with yak fat differentiation has not been reported,and its gene sequence is unknown,which restricts the related functional research.In order to obtain the conserved region sequence of yak MAP3K7 gene coding region (CDS),clarify its expression characteristics,and understand its expression level in different tissues of yak and its correlation with subcutaneous preadipocyte differentiation of yak,the MAP3K7 gene of yak was cloned by RT-PCR and the biological characteristics were analyzed by bioinformatics methods.Real-time Fluorescence Quantitative PCR (qPCR) was used to detect the expression of MAP3K7 in 10 tissues of yak heart,liver,spleen,etc,and the expression of MAP3K7 gene and lipid differentiation-related genes in yak subcutaneous adipocytes at different differentiation stages.At the same time,combined with oil red O staining,the effect of MAP3K7 gene on the differentiation of yak subcutaneous preadipocytes was clarified.The results showed that the length of the CDS region of the cloned yak MAP3K7 gene was 1 740 bp,encoding 579 amino acids.Compared with the CDS region of the wild yak MAP3K7 predicted sequence,it was found that A>G at position 967,resulting in a mutation of Thr>Ala at position 323.The analysis of physical and chemical properties showed that the protein was a negatively charged hydrophilic acidic protein with 112 amino acid phosphorylation sites.Among its amino acid composition,serine accounted for the highest proportion.The secondary structure prediction showed that the content of random coil was the highest,which might interact with proteins such as TAB3.The results of nucleotide homology comparison and phylogenetic tree showed that yak MAP3K7 was highly conserved among ruminants,and had the highest homology and the closest evolutionary relationship with wild yak.Tissue quantitative detection showed that the expression of MAP3K7 in subcutaneous fat and visceral fat of yak was relatively low.During the differentiation of subcutaneous preadipocytes of yak,the expression of MAP3K7 was up-regulated with the increase of mature adipocytes,and then down-regulated after differentiation to a certain extent.In summary,MAP3K7 gene was highly expressed in the early stage of differentiation of yak subcutaneous preadipocytes to promote differentiation,but when the differentiation reached a certain level,the down-regulation of expression inhibited further differentiation.

The tail type of sheep is a complex trait formed by the interaction of genetic and environmental factors.circRNA is closely related to lipogenesis.To investigate the effect of circular RNA(circRNA)on the tail fat deposition of sheep,transcriptome sequencing and differential expression analysis of sheep tail fat were performed.Candidate circRNA associated with sheep tail fat were screened,and the regulatory network diagram of circRNA-miRNA-mRNA associated with sheep tail fat deposition was constructed,the selected circRNA were located,and their functions were verified.The results showed that a total of 679 differentially expressed circRNA were detected in the transcripts of adipose tissue of two different tail types of sheep,of which 422 were up-regulated and 257 down-regulated.Moreover,GO and KEGG functional enrichment analysis was performed on differentially circRNA target genes,which involved many biological development processes such as DNA metabolism,anatomical structure development,catabolic process,autophagy,carbohydrate absorption process,cell proliferation and lipid metabolism related to fat deposition.Target gene enrichment was involved in cell growth and apoptosis,cell motility,transport and catabolism,signal transduction,transcription and translation,amino acid anabolism and other functions,suggesting that these circRNA may participate in the deposition process of sheep tail fat through the above pathways.The selected differential circRNA_0018 was localized by fluorescence in situ hybridization and verified in the precursor adipocytes.The results showed that circRNA_0018 was a true and stable cytoplasmic ring molecule,and functional verification of circRNA_0018 showed that it could promote adipocyte differentiation.circRNA_0018 may be involved in the process of fat deposition and lipid metabolism in sheep.

To analyze the variability of the novel Goose astrovirus(GAstV)in Lu'an,Anhui Province and to express the VP27-VP34 fusion protein,the gout samples were collected from a farm in Lu'an.After confirming positivity via RT-PCR,the virus was isolated by passage culture in goose embryos.Then,the isolated strain was subjected to an animal regression test,whole genome amplification sequencing and genetic evolution analysis.Subsequently,the VP27-VP34 fusion protein of the isolated strain was induced and expressed,and purified recombinant protein was used to immunize 6-week-old female BALB/c mice to produce polyclonal antibodies.Serum antibody titers were assessed using agar diffusion methods,and the specificity of the polyclonal antibodies was detected by indirect immunofluorescence(IFA).The specificity of the antibody was detected by indirect immunofluorescence(IFA),and the titer of the prepared antibody was detected by the agar diffusion method.The results showed that one strain of GAstV,named AH-2021 strain,was isolated from clinical samples.The animal regression test showed obvious urate deposition on the surface of the heart and liver of goslings,and the kidney was white and swollen.Genetic evolution results revealed that AH-2021 belonged to GAstV-1,showing 98.0%—99.0% identity with other GAstV-1 strains in GenBank.The recombinant expression vector pCold-TF-VP27-VP34 was induced by IPTG to obtain the target protein,and SDS-PAGE showed that the molecular weight of the recombinant protein was about 110 ku,which mainly existed in the form of supernatant.IFA results showed that the polyclonal antibody was able to specifically recognize the GAstV,and the agar diffusion results showed that the titer of polyclonal antibody was up to 1:16.In conclusion,a strain of novel GAstV AH-2021 was isolated from gouty goslings,and animal regression tests showed that the novel Goose astrovirus was the pathogen causing gout in goslings,and a polyclonal antibody to the VP27-VP34 fusion protein was prepared.

Elongation of very long chain fatty acids protein 3(ELOVL3),mainly involved in the synthesis of very long chain fatty acids(VLCFA),plays an important role in fatty acid metabolism.The objectives of this study were to clone the protein-coding sequence of ELOVL3 gene from Jiulong yak,to construct the eukaryotic expression vector of ELOVL3 gene,and to predict and analyze its biological functions.The CDS region of the ELOVL3 gene was amplified by PCR and inserted into the pcDNA3.1 vector by homologous recombination to construct a recombinant plasmid.The recombinant plasmid was validated by restriction digest and PCR,and the biological functions of its protein coding sequence were analyzed by online prediction software in combination with sequencing results.In addition,qPCR and Western Blot were performed for the expression of ELOVL3 gene at mRNA level and protein level.The results showed that the protein-coding sequence of Jiulong yak ELOVL3 gene was 813 bp in length,encoding a total of 270 amino acids.Enzymatic cleavage,PCR and sequencing identification confirmed the successful construction of the pcDNA3.1-ELOVL3 eukaryotic expression vector.Bioinformatics analysis showed that the protein encoded by the ELOVL3 gene was a hydrophobic protein,with 6 transmembrane structural domains and 27 phosphorylation sites.The phylogenetic tree showed that the closest relation to the Jiulong yak was the common cow,and the furthest was the original chicken.In addition,the ELOVL3 gene was most highly expressed in the lung tissue of the Jiulong yak.

In order to investigate the expression pattern and biological function of lncRNA TCONS_00143126 in E.coli type mastitis of cows in depth.This study used cDNA from bovine mammary epithelial cells as a template,and confirmed the presence of lncRNA TCONS_00143126 using PCR cloning and sequencing techniques.Subcellular localization analysis of lncRNA was performed,and potential target miRNAs and genes were predicted.The potential mechanism of its action in bovine mastitis was explored through KEGG pathway enrichment analysis.In addition,LPS was used to induce bMECs to construct an in vitro model of bovine mastitis,and the expression of lncRNA TCONS_00143126 in LPS-induced bMECs at 6,12 and 24 h was detected by RT-qPCR.The results showed that lncRNA TCONS_00143126 was real,and its expression was significantly up-regulated in LPS-induced bMECs,and it was mainly distributed in the nucleus.The results of target gene prediction and KEGG enrichment analysis showed that lncRNA TCONS_00143126 might regulate inflammatory signaling pathways such as JAK-STAT,mTOR and MAPK by targeting miRNAs(bta-miR-133a,bta-miR-193a-5p and bta-miR-375,etc.)and target genes(IFNE,SLC2A10,MEX3B),and then play a role in the inflammation of bovine mammary epithelial cells.

Anoctamin 5(ANO5)is a multichannel membrane protein localized in the sarcoplasmic and sarcoplasmic reticulum that primarily plays a role in myosin membrane repair and phospholipid scrambling,mutations in the ANO5 gene can lead to jaw hypoplasia as well as various myopathies.It aimed to investigate the association of SNPs in the ANO5 gene with fat deposition traits in sheep.A population of 1 005 healthy and clearly genealogical Hu sheep male lambs was selected for the study,and PCR amplification and KASPar typing techniques were used to detect the locus polymorphisms of the ANO5 gene in the experimental population and analyze the associations with fat deposition traits.The expression level of ANO5 gene in different tissues was analyzed by qPCR.The results showed that sheep ANO5 gene was widely expressed in a variety of tissues in Hu sheep,and the highest expression of ANO5 gene was found in heart tissue compared with other tissues.Three genotypes of CC,CT and TT with the g.58010 C>T polymorphic locus were detected in the 10th intron of the sheep ANO5 gene.Descriptive statistics showed that the perirenal fat weight was the most different and had the highest degree of variability compared with other fat weights.Correlation analysis showed that fat deposition related traits were positively correlated with growth and feed efficiency traits,and the results of association analyses showed that the polymorphic locus was significantly associated with perirenal fat weight and its related traits in the Hu sheep.Among them,the perirenal fat weight of individuals with CC genotype was significantly lower than that of individuals with TT genotype.In conclusion,the g.58010 C>T mutation locus of the ANO5 gene can be used as a candidate molecular marker for perirenal fat deposition traits in Hu sheep.

This study aimed to compare and analyze the milk composition,serum biochemical indices and expression levels of Leptin and LF genes during lactation between Bamei pigs and Yorkshire pigs.The six purebred Bamei pigs and Yorkshire pigs with healthy body conditions were selected respectively,which also had close parity,mating and delivery periods(within three days).A total of 50 mL milk,2 mL serum and 2 mL whole blood samples were collected from Bamei pigs and Yorkshire pigs on the 1st,7th,14th,21st and 28th day after delivery,respectively.Then,the milk,serum and whole blood samples were used to detect the milk compositions,serum indexes of alkaline phosphatase(ALP),albumin(ALB),globulin(GLB),triglyceride(TG),urea nitrogen(BUN),alanine aminotransferase(ALT)and aspartate aminotransferase(AST),and the expression levels of Leptin and LF genes.The results showed that on the 1st day of lactation in Baimei pigs,in addition to milk fat(Fat),the content of casein(CS),total protein(TP),total solid matter(TS),non-fat milk solids(SNF),lactose(L),free fatty acid(FFA)and acidity in the colostrum of Bamei pigs were all extremely significantly higher than those in the colostrum of Yorkshire pigs,while,the content of CS,TP,Fat,TS,SNF and L of Bamei pigs on the 21st day after delivery were extremely significantly lower than those of Yorkshire pigs.The serum biochemical indexes of two pig breeds showed different trends during the whole lactation.The content of ALT,AST,GLB,BUN and TG of Bamei pigs were significantly higher than those of Yorkshire pigs on the 1st day after delivery,while the content of ALP and ALB were all significantly lower than those of Yorkshire pigs.There were no different levels of these two genes on the 1st day,however,from the 7th day after delivery,the expression levels of LF in the Bamei pigs were significantly different than those in the Yorkshire pigs,while Leptin gene was extremely significantly expressed in Yorkshire pigs than in Bamei pigs.In general,the milk nutrients of Bamei and Yorkshire pigs showed a decreasing trend as the extension of the lactation periods,and the expression levels of Leptin and LF genes were likely related to the milk yield and quality between the two breeds,which may need to be further verified.

The purpose of this study is to screen candidate genes related to the body weight at 8 weeks age of Kangle yellow chicken based on the omics data,and provide the theoretical foundation for molecular marker-assisted breeding of growth traits in Kangle yellow chickens.It also provides key basic data for improving molecular breeding methods for high-quality broilers and accelerating,the progress of breed selection.To detect SNPs significantly associated with body weight traits at 8 weeks age of Kangle yellow chickens,the body weight was measured from 8 to 22 weeks of age of 434 Kangle yellow chickens.Genome-wide association study(GWAS)was performed using the gene chip technology.Genes in the candidate regions with 2 Mb windows surrounding each significant SNP were found for GO and KEGG function analysis.Combined with the data of transcriptomic sequencing and published literature,key candidate genes related to the body weight at 8 weeks age of Kangle yellow chicken were screened.Two potential SNPs significantly associated with target traits were detected,located on chromosome 2(131 485 613 bp)and chromosome 4(60 413 848 bp).A total of 118 candidate genes were screened near SNP sites.Gene function annotation analysis showed the most significant enrichment of biological processes was retinoic acid metabolism.The significant enrichment of 12 KEGG pathways was found,including fatty acid degradation,tyrosine metabolism and drug metabolism-cytochrome P450.OXR1, RSPO2,EIF3E,TRHR,BMPR1B, ADH1C, MTTP, LAMTOR3, PPP3CA and PDLIM3 were preliminarily identified as key candidate genes for body weight traits at 8 weeks age of Kangle yellow chickens.Two SNPs and 10 key candidate genes were preliminarily identified related to the body weight at 8 weeks age of Kangle yellow chicken.

In order to explore the sequence characteristics of yak Akirin1 gene and understand its tissue expression characteristics in detail.Gannan yak muscle tissue cDNA was used as a template to clone the CDS sequence of Gannan yak Akirin1 gene by RT-PCR technology,and bioinformatics analysis was performed using a variety of biological online software and tools.The qPCR technique was used to detect the difference expression of Akirin1 gene in Gannan yak heart,liver,spleen,lung,kidney,muscle,fat and testis tissues.The results showed that the CDS of Akirin1 gene in Gannan yak was 579 bp,encoding 192 amino acids.The protein molecular formula encoded by the gene was C₉₆₄H₁₅₂₅N₂₇₅O₂₉₁S₈,the theoretical isoelectric point of the protein was 8.91,the instability coefficient and the total average hydrophilicity were 84.14 and -0.822,respectively,which belonged to the unstable hydrophilic protein.There were 29 potential phosphorylation sites,including 14 serines,11 threonines and 4 tyrosines.There was no signal peptide and transmembrane helix structure.Subcellular localization showed that it mainly existed in the nucleus and belonged to non-secretory protein.The high-level structure of the protein was mainly composed of 39.06% α-helix and 56.25% random coil structure.Homology analysis showed that the genetic relationship between yak and cattle was the closest,which was in line with the actual situation,indicating that the Akirin1 gene was relatively conservative in the evolution process.Real-time Fluorescence Quantitative results showed that Akirin1 gene was expressed in heart,liver,spleen,lung,kidney,muscle and adipose tissue of adult Gannan yak,and the relative expression in kidney tissue was the highest,which was significantly higher than that in other tissues,and the expression in muscle and liver tissue was relatively low.This experiment provides a theoretical basis for further study on the functional characteristics of yak Akirin1 gene.

The aim of this study was to establish yak muscle and skin fibroblast cell lines,analyze and compare the differences in morphology,growth,marker genes and proteins between two cell lines to provide materials for a better understanding of yak properties.Firstly,the skin and muscle tissue blocks were isolated from the yak fetus at 3 months of gestation,and the primary cells were cultured by tissue block method and enzymatic hydrolysis method respectively,and their growth status and morphological characteristics were observed.Following,the CCK8 method to detect the proliferation viability of skin and muscle fibroblasts,and immunofluorescence staining analysis was performed on the two cells.Finally,RT-qPCR was used to analyze the expression of fibroblast marker genes in the two kinds of cells.It was found that the enzymatic method was easier to obtain fibroblasts than the tissue method,and the viability of skin fibroblasts was significantly higher than that of muscle fibroblasts(P<0.05).The fluorescence intensity of the marker protein VIMENTIN in skin fibroblasts was significantly higher than that of muscle fibroblasts,but the histone-modified H3K27me3 was not significant between the two kinds of cells.Furthermore,VIMENTIN and S100A4 genes were significantly highly expressed in skin fibroblasts,while FAP and SMA genes were significantly high expressed in muscle fibroblasts,consisting with immunofluorescent staining results.In conclusion,it successfully established yak fetal skin and muscle fibroblast cell lines,and compared their physiological properties of two kinds of cells by using CCK8,immunofluorescence staining and RT-qPCR.

To investigate the infection of BEFV and the genetic diversity of its prevalent strain G gene in a dairy farming community in Dali Prefecture,Yunnan Province.It designed and synthesized two pairs of specific amplified G genes for 10 suspected cases in dairy cattle breeding community of Dali Prefecture,with reference to the G gene sequence of bovine ephemeral fever virus in GenBank,sequenced and completed sequence analysis.At the same time,60 serum samples were collected for antibody detection.The results showed that 6 out of 10 whole blood samples of cattle suspected of BEF were positive for BEFV nucleic acid.The G gene of four bovine ephemeral fever virus Yunnan epidemic strains was amplified and sequenced by removing the repetitive sequence.The total sequence length was 1 872 bp,encoding 623 aa.The nucleotide sequence homology among the four strains was 99.7%-99.9%.Phylogenetic analysis showed that the four strains were grouped together with Asian strains and were a small branch alone.It had the closest relationship with TH-NP0065 strain isolated from Thailand in 2017 and Chinese strain.Compared with the amino acid homology of the vaccine strain JB76H in China,seven amino acid mutation sites were found:K⁵³ → N⁵³,K⁶² → R⁶²,P¹⁸³ → S¹⁸³,G²⁶³ → E²⁶³,K⁴⁶¹ → E⁴⁶¹,K⁴⁵⁷ → R⁴⁵⁷,I⁴⁷⁵ → M⁴⁷⁵.Except the neutralizing antigen site G1,mutation sites were present in G2,G3 and G4.According to the results of antibody test kit,19 samples with antibody concentration≥52.5 ng/L critical value were found,with a positive rate of 31.67%.In conclusion,BEFV infection was present in this dairy farming community and there were multi-locus variants in the G gene of the prevalent strain.

The aim of this study was to explore the mechanism of flavor difference of Angus beef at different months of age based on metabonomics analysis.Angus longissimus dorsi muscle of 17,20 and 24 months old was tested for meat quality traits and metabolomics analysis under the same feeding and management conditions.The metabolome results showed that 69,51 and 53 positive differential metabolites and 53,46 and 58 negative differential metabolites were identified in the B17 vs B20, B17 vs B24 and B20 vs B24 groups,respectively.The expression of 166 differential metabolites was up-regulated and 164 differential metabolites were down-regulated.KEGG enrichment analysis showed that differential metabolites were mainly enriched into multiple metabolic pathways such as lipid metabolism,amino acid metabolism,carbohydrate metabolism and endocrine system,including insulin resistance,cholinergic synapse,linoleic acid metabolism,phosphopentose pathway,carbohydrate digestion and absorption,glucagon signaling and arachidonic acid metabolic pathway.Correlation analysis showed that key metabolites such as proglutathione,betaine,carnitine,choline,hypoxanthine,palmitoleic acid,citric acid and lactose were significantly correlated with phenotypic data and could be used as potential candidate metabolites for Angus beef.Our results indicate that there are significant differences in the quality and flavor of Angus beef at different months of age,and the identified differences in metabolites and metabolic pathways can provide a theoretical basis for future Angus cattle breeding.

bta-miR-6523a is one of the genes related to bovine fat deposition screened by the previous research Shi Yuangang's group through sequencing data.In order to screen the target gene of bta-miR-6523a,identify its expression rule in different tissues,and explore the possible regulatory mechanism of bta-miR-6523a on bovine fat deposition,this study used a variety of database information to predict and screen the target gene of bta-miR-6523a,and enriched and analyzed the predicted functional pathway of the target gene.Finally,the expression of bta-miR-6523a in some tissues of adult cattle and calves was detected by qRT-PCR.The results showed that the predicted target genes of bta-miR-6523a were SCD,CIDEA,LIPE,etc.These predicted target genes were significantly enriched in Ras,Rap1,actin cytoskeleton regulation,Hippo and other signaling pathways related to adipocyte generation and differentiation,cytoskeleton organization and maintenance,and metabolic pathways.qRT-PCR results showed that the expression level of bovine miR-6523a was the highest in lung and the lowest in perirenal fat.The expression of bta-miR-6523a in heart and liver was significantly higher than that in subcutaneous fat,intermuscular fat,longissimus dorsi muscle and perirenal fat,but there was no significant difference among subcutaneous fat,intermuscular fat,longissimus dorsi muscle and perirenal fat.The expression of miR-6523a in subcutaneous fat,intermuscular fat and longissimus dorsi muscle of calves was significantly different,and significantly lower than that in adult bovine tissues.The high expression of bta-miR-6523a in lung tissue and lower expression in adipose tissue suggest that the biological effects of bta-miR-6523a and its target genes may be extensive.

In order to grasp the genetic characteristics of sheep tail length trait,evaluate the genetic effect of c.C334A genotype of TBXT gene,a candidate marker related to sheep tail length variation,and provide a reference for breeding sheep short tail trait.The long-tailed Texel sheep,tailless Kazakh sheep,and hybrid F₁ and F₂ progeny populations were selected as the research objects.The genetic pattern of the number of tail vertebrae was analyzed,and the correlation between the number of tail vertebrae and tail length was analyzed.The genotype effect of TBXT c.C334A on tail length variation was evaluated in the hybrid population of Texel sheep and Kazakh sheep using SPSS 26.0 regression analysis.The results showed that the average number of caudal vertebrae,skewness and peakedness of Texel sheep were 17.87±1.30,1.18±0.58 and 1.37±1.12,respectively.The average number of caudal vertebrae,skewness and peakedness of Kazak sheep were 3.52±1.03,0.54±0.50 and 0.34±0.97,respectively.The average number of caudal vertebrae,skewness and peakedness of the F₁ population were 12.61±1.82,-0.40±0.54 and 1.52±1.08,respectively.The average number of caudal vertebrae,skewness and peakedness of the F₂ population were 11.67±2.63,-0.70±0.23 and-0.12±0.46,respectively.It showed that the inheritance of the number of sheep tail vertebrae in parents and offspring conformed to the genetic pattern of quantitative traits.The coefficient of determination R² of the number of tail vertebrae to the tail length of sheep was 0.726,indicating that the change of the number of tail vertebrae was the main influencing factor of the tail length variation.The additive effect,dominant effect and substitution effect of TBXT c.C334A on tail length variation were 2.62,0.14 and 2.57,respectively.This locus explained 43.96% of the phenotypic variation of tail length,suggesting that TBXT c.C334A is a genetic marker that affects the tail length by affecting the number of tail vertebrae.

The aim of the study was to investigate the stress changes in the intestinal tract of piglets after weaning and the effect on the expression of AA transporter carriers in the intestinal mucosa of Wuzhishan pigs.A total of 24 Wuzhishan piglets were used, which were slaughtered on 0 days of weaning,and 3,7 and 10 days after weaning, and serum, small intestinal segments and mucosa were collected. Their blood immunity indexes,small intestine morphology and intestinal mucosa AA transporter carrier-related gene expression were determined.The results showed that compared with the pre-weaning period,the intestinal villi were sparsely broken,the duodenal and ileal villi heights were reduced in the 3rd day after weaning,and the differences in the changes in the surface area of jejunal and ileal villi were significant.All the values of villi height improved after the 7th day after weaning; the content of serum total protein,albumin,globulin and glucose decreased significantly after weaning,and the differences in aspartate aminotransferase,total cholesterol,lactate dehydrogenase and aspartate aminotransferase content were significant; the changes in serum diamine oxidase,D-lactic acid and lipopolysaccharide were significant after weaning; the relative expression abundance of the small peptide transporter PepT1 increased in the order of duodenum,jejunum and ileum after weaning,the relative expression abundance of the acidic AA transporter EAAT3 was not significant in the intestine,the relative expression of the neutral AA transporters B⁰AT1,ASCT2,and rBAT was higher in the jejunum as well as in the ileum,and the basic AA transporters CAT1,y+LAT1 and 4F2hc,had reduced expression in the duodenum after weaning,and the vectors were relatively more stable in ileal expression.In summary,early weaning stress caused obvious intestinal changes such as shortening of villi and crypt hyperplasia in Wuzhishan piglets,and decreased the content of blood immunoproteins and diamine oxidase,D-lactic acid and lipopolysaccharides,etc.At the same time,the expression of intestinal AA transporter carriers was changed,especially in the 3rd day of the post-weaning period,and all the indexes changed significantly,but with the growth of weaning day old up to the 10th day of the weaning period,it gradually recovered,and the piglets' immunity increased,and the permeability of the intestinal mucosa increased.

In order to investigate the regulatory level of PLA2G4A gene on inosine monophosphate(IMP)in the leg muscle tissue of Bashang Long-tailed chicken,and further explore the biological function of PLA2G4A gene in Bashang Long-tailed chicken.The leg muscle tissue of 270-day-old Bashang Long-tailed chicken was categorized based on the inosine monophosphate content,with five samples allocated to each group.Transcriptome sequencing analysis was conducted on the muscle tissue,followed by KEGG enrichment analysis,GO function annotation and protein interaction network analysis.RT-qPCR was used to analyze the mRNA expression level gene in the leg muscle tissue of Bashang Long-tailed chicken.KEGG enrichment analysis showed that the signal pathways related to IMP synthesis included glycerophospholipid metabolism,etherlipid metabolism and arachidonic acid metabolism.GO functional annotation showed that the PLA2G4A gene was involved in 11 biological processes,including glycerophosphate metabolism,etherlipid metabolism,arachidonic acid metabolism and linoleic acid metabolism.Protein interaction revealed that the PLA2G4A gene shared important regulatory enzyme functions in arachidonic acid metabolism and glycerophosphate metabolism.The Real-time Fluorescence Quantitative PCR results showed that there was a significant difference in the expression level of the PLA2G4A gene in the leg muscle tissue of Bashang Long-tailed chicken in the sample.In summary,the PLA2G4A gene may be the main gene affecting IMP deposition in chicken.

The transcriptional regulation of spleen tissue at different time points after Salmonella infection in Muscovy ducks was analyzed by transcriptome sequencing technology,to investigate the impact of Salmonella infection on Muscovy ducks.Fourteen-day-old Muscovy ducks were infected with Salmonella enteritidis.After 1,3,and 10 days of infection,spleen tissue samples were collected from the experimental and control groups,and RNA was extracted.The Illumina sequencing platform was used for transcriptome sequencing,and changes in transcriptome,signaling pathways,and functions at different time points were identified through bioinformatics analysis.The results showed that 95,53,and 12 differential genes(P<0.05,| log₂(fold change)|>1)were found in Muscovy ducks infected with Salmonella for 1,3 and 10 days,respectively.GO analysis showed that these differential genes were enriched in biological processes,molecular functions,and cellular components at the three time points.KEGG analysis showed that at 1 day of infection,the most differentially expressed genes of CXCR4,CCL3,GDF9 and CNTFR were enriched in the cytokine-cytokine interaction signaling pathway.During the third day of infection,the differentially expressed genes of ADRA1A,ADRA1b,GRIA1,and MC5R were enriched in the neural active ligand receptor interaction signaling pathway;after 10 days of infection,the differentially expressed gene H2-EB1 participates in multiple signaling pathways,indicating that these signaling pathways and differentially expressed genes may play an important role in the infection process of Salmonella in Muscovy ducks.

Investigating the predominant genotypes of Bovine viral diarrhea virus(BVDV)infecting cattle in Tongliao,Inner Mongolia,to provide reference for the BVDV epidemiology and prevention and control.In the preliminary phase of the experiment,fecal samples from diarrheic calves were collected at a cattle farm in Tongliao,Inner Mongolia.These samples were tested using PCR to detect BVDV positivity.Positive fecal samples were then inoculated into madin-darby bovine kidney cells(MDBK)for isolation.The isolated strains were identified using RT-PCR and indirect immunofluorescence staining.Subsequently,the full-length genome of the isolates was sequenced,followed by genetic evolution analysis and genotype determination based on sequences of the 5'UTR,N^pro,and E2 genes.The results indicated that this experiment successfully isolated a strain of BVDV,designated as NM-21.Inoculation of NM-21 into MDBK did not induce cytopathic effects,indicating it was a non-cytopathic strain(NCP).Both RT-PCR and indirect immunofluorescence staining confirmed its positivity,with a virus titer of 10^-3 TCID₅₀/mL.Based on the full-length genomic sequence,and homology and genetic evolution analysis of the 5'UTR,N^pro,and E2 gene sequences,the isolate NM-21 showed the highest nucleotide homology with the BVDV-1c subtype strain NM2103(GenBank accession number ON337882.1)from Inner Mongolia,China.

To study the structure and function of phosphotyrosine interaction domain 1 (PID1) gene in yak,and to explore its expression in various tissues.The CDS region of Sangsang yak PID1 gene was cloned using Sangsang yak adipose tissue cDNA as template,and the sequence was analyzed bioinformatically.Meanwhile,the relative expression level of PID1 gene was detected in seven tissues of yak namely heart,liver,spleen,lung,kidney,longissimus dorsi muscle and adipose tissue by Real-time Fluorescence Quantitative PCR(RT-qPCR).The results showed that the PID1 gene in yaks had a coding region length of 654 bp,which encoded 217 amino acids.Homology comparison showed that yak and wild yak were closely related,and the similarity reached 100%.The molecular weight of yak PID1 protein was about 24.84 ku and the theoretical isoelectric point was 6.30.According to the calculation results of instability coefficient,the instability of the protein was high (47.96),and it belonged to an unstable protein.The protein had one N-glycosylation site and 23 phosphorylation sites,with no signal peptide or transmembrane structure.The results of RT-qPCR showed that the expression of PID1 gene could be detected in all tissues,with the highest expression in the lung.The yak PID1 gene was cloned and its protein structure was analyzed.The expression of PID1 gene in yak tissue was also studied.Further study on the role of PID1 gene in yak fat deposition provided preliminary data.

The highly contagious gastrointestinal infectious disease caused by Porcine epidemic diarrhea virus(PEDV)has led to significant economic losses in China's pig industry.It aimed to establish a basis for PEDV antibody detection methods and functional research of the N protein through the screening of specific nanobodies(Nbs)using phage display technology.Peripheral blood lymphocytes were isolated from a camel immunized with the N protein,and total RNA was extracted and reverse transcribed into cDNA.The variable domain of the heavy chain of heavy chain antibodies(VHH)was amplified by PCR,subcloned into the pCANTAB5E-ccdb vector,and electroporated into ER2738 competent cells to construct the VHH phage antibody display library.Subsequently,the library was subjected to four rounds of panning against the PEDV N protein,and positive phage clones were cloned into the pET-30a vector.The binding affinity and specificity of the Nbs were determined by indirect ELISA and Western Blot.The results showed that after the fifth immunization,the antibody titer reached 1∶25 600.The constructed phage display library had a capacity of 4.72×10⁸ and an abundance of 4.3×10¹⁰ cfu/mL,with a 93.75% positive rate.After four rounds of screening,16 Nb clones with different amino acid sequences were obtained,and Nb45 was validated to possess excellent specificity and binding ability to the PEDV N protein.This study successfully screened and obtained specific N protein-targeting Nbs,providing biological materials for the establishment of PEDV detection methods and foundational research.

The aim of this study is to analyze the structure and biological function of ORF2 protein of Avian hepatitis E virus (aHEV)CaHEV-GDSZ01 strain,and the recombinant ORF2 protein was expressed in E.coli prokaryotic expression system and polyclonal antibodies against ORF2 protein were prepared to verify the immunogenicity and reactivity of the recombinant protein.The physicochemical properties,structure and function of ORF2 proteins of CaHEV-GDSZ01 strain were analyzed by bioinformatics software.The prokaryotic expression plasmids of pET-32a-ORF2-His and pET-32a-ORF2 were constructed by digesting and ligating pET-32a(+)vector with ORF2 gene of CaHEV GDSZ01 strain,and then transformed into DE3 competent cells for induction and expression,and the expression form was analyzed.The recombinant ORF2 protein was purified by gel cutting,its reactivity was verified by Western Blot test,and then rabbits were immunized to obtain rabbit anti-ORF2 protein polyclonal antibody.The polyclonal antibody was identified and analyzed by Western Blot,and the antibody titer was detected by indirect ELISA.The results of bioinformatics analysis showed that the ORF2 protein of CaHEV-GDSZ01 strain was hydrophilic and unstable,and the probability of the existence of signal peptide was as high as 93.59%.The secondary structure of the protein was mainly irregular curl and had the structural conditions for binding antibodies.And the overall prediction score of protective antigen of ORF2 protein showed that it had good immunogenicity.ORF2 gene prokaryotic expression plasmids was successfully constructed,and the target protein was successfully expressed.SDS-PAGE showed that the protein was expressed as insoluble protein,and the protein was proved to have good reactivity by Western Blot.The titer of the prepared polyclonal antibody was 1∶102 400,and it could specifically recognize immunogen ORF2 protein,non-immunogen ORF2-His protein and truncated expressed sORF2 protein.The physicochemical properties and some biological functions of ORF2 protein was compared and analyzed,the related proteins were expressed successfully,and the rabbit polyclonal antibody against ORF2 protein was prepared.

In order to analyze the possible role of dTLR2 in duck innate immunity,we study the expression of dTLR2 mRNA in immune organs during 1 to 10 weeks,as well as the expression in blood after challenging with Escherichia coli and Salmonella enteritis by Fluorescence Quantitative PCR.We also prepared polyclonal antibodies of the extracellular domain of dTLR2 by recombinant protein expression for further studying the biological function of dTLR2.The results showed that dTLR2 mRNA was expressed in spleen,thymus and bursa at different weeks.The expression of dTLR2 mRNA was significantly different among different weeks in the spleen and thymus tissues,and there was no significant difference in bursa.After infection with E.coli and S.enteritis,the expression of dTLR2 mRNA was significantly higher than that of the control group in the first and second days,but there was no significant difference among groups in the third day.Analyzing the sequence of dTLR2 gene,we predicted the extracellular domain,designed primers for PCR assay,and built pET28a-TLR2 recombinant plasmid.The recombinant plasmid was translated into E.coli BL21 (DE3) for expression of dTLR2-His protein.The SDS-PAGE results showed that the fusion recombinant protein efficiently expressed in the supernatant,molecular mass was around 29 ku.The results of Western Blot showed that the antibody which harvested from immunizing rabbits could detect recombinant dTLR2 and endogenous dTLR2.The analysis of the expression of dTLR2 and successful preparation of polyclonal antibody provide favorable support for further studying of dTLR2 biology function.

This study aimed to detect the SNPs of CDKN2A gene in Wenshang Barred chicken,and perform the analysis of its bioinformatics and expressions in skin tissues.Based on the high quality chromosome-level genome of Wenshang Barred chicken assembled by the research group of Resource Conservation and Evaluation in Jiangsu Institute of Poultry Science,the sequences were blasted to chicken reference genome (GRCg7b) and the SNPs were acquired.These SNPs were detected and genotyped by MassARRAY nucleic acid mass spectrometry in the chicken population.Bioinformatics software was used to predict the regulatory sequence of CDKN2A gene.The physicochemical properties and structural characteristics of the encoded protein were analyzed.The expressions of this gene in the skin tissues of Wenshang Barred chicken at different periods were detected by transcriptome and Real-Time Fluorescence Quantitative PCR.The results showed that the total length of CDKN2A and its CDS were respectively 9.854 kb and 183 bp,encoding 60 amino acids.Forty-one SNPs were found in this gene and its upstream and downstream 1 kb,of which four SNPs were newly discovered loci.These four new SNPs were located in the downstream and the intron,respectively.The first exon had two SNPs,both of which were missense mutations:V9D and C10R.The detection rates of 34 SNPs among the 41 SNPs were all over 91% in the Wenshang Barred chicken population.These SNPs were almost homozygous mutants without polymorphism.Eight promoters and five CpG islands were predicted in the 2 kb upstream of CDKN2A in Wenshang Barred chicken.The promoter region contained a total of 12 transcription factor binding sites,including Sp1,MEB-1,YY1,C/EBPα,AP-2α.The protein encoded by this gene was a positively charged,unstable hydrophilic protein with a molecular mass of 7.30 ku and a theoretical isoelectric point of 12.82.The predictive subcellular localization of the protein was mainly in mitochondria.The protein had no signal peptide regions and signal peptide shear sites.It had eight potential phosphorylation sites and no glycosylation sites.The protein contained a typical TRP_2 (transient receptor potential ion channelⅡ) conserved domain and its secondary and tertiary structures were mainly α-helices and random coil.In addition,it had interactions with MDM2,NPM1,USP36 proteins.The expressions of CDKN2A gene in the back skins increased from 1 to 35 days old in Wenshang Barred chicken.

The aim of this study was to investigate the correlation of single nucleotide polymorphisms of the carboxyl ester lipase gene (CEL) with eye muscle area in Hu sheep.The 1 049 Hu sheep with accurate phenotypic records and in good health were selected,their eye muscle area was determined after slaughter,and blood was collected for genomic DNA extraction.Amplification of target fragments and genotyping of CEL gene by PCR and KASPar SNP typing,and association analysis with eye muscle area of Hu sheep,RT-qPCR was used to detect the expression of CEL gene in 10 tissues of Hu sheep.The results showed that the Hu sheep CEL gene was expressed in different tissues and was highest in the duodenum.There was a synonymous mutation of g.4039718 C>T in this gene,which was moderately polymorphic.Trait association analysis showed that this polymorphic locus was significantly associated with eye muscle area in Hu sheep,with TT-type individuals having significantly higher eye muscle area than CC-type individuals.Descriptive statistics showed that the coefficient of variation for eye muscle area was 12.29%,which had high selection potential.Correlation analysis showed that eye muscle area was significantly and positively correlated with growth and slaughter traits such as body weight,body height,breast circumference,slaughter rate,carcass weight and live weight before slaughter.The results indicated that the g.4039718 C>T polymorphic locus could be used as a candidate molecular marker for genetic improvement of eye muscle area traits in Hu sheep.

To investigate the regulatory effect of DEAD-box helicase 21(DDX21)on the replication of Transmissible gastroenteritis virus(TGEV).Firstly,Western Blot was utilized to analyze the effect of TGEV infection on DDX21 expression.Furthermore,we constructed eukaryotic expression plasmids of porcine DDX21 and established knockdown stable cell lines.RT-qPCR,Western Blot,Indirect immunofluorescence(IFA)and TCID₅₀ assays were used to investigate the regulatory effect of DDX21 on TGEV replication in vitro.Western Blot analysis showed that the protein levels of DDX21 were significantly up-regulated in PK-15 cells at the early stage of TGEV infection.RT-qPCR,Western Blot,IFA and TCID₅₀ experiments showed that over-expression of DDX21 significantly increased the mRNA level and protein of TGEV N in a dose-dependent manner.And the amino acids 601—784 aa of DDX21 were critical for promoting TGEV replication.Otherwise,the titer of TGEV was significantly down-regulated in DDX21 knockdown cell lines,whereas the titer of TGEV in DDX21 knockdown cell lines was reversed under the rescue experiment.This study revealed for the first time that DDX21 promotes the proliferation of TGEV and identified the key domain of DDX21 in regulating TGEV replication,which provided a theoretical a basis for future research on the function of DDX21 protein and the pathogenesis of TGEV.

Epidermal growth factor receptor(EGFR)is excessively expressed in numerous solid tumours,establishing a stable over-expressing canine EGFR protein MDCK cell line will prove beneficial in providing an exceptional cellular model and research foundation for the study of canine or human tumour diseases.Firstly,the target fragment was prepared for construction of an overexpression vector of the EGFR gene.It was co-transfected with a lentivirus packaging vector into 293T cells.After 48 hours of transfection,the supernatant was extracted and purified to obtain a lentivirus containing the overexpressed EGFR gene.This virus was then transduced into MDCK cells,following puromycin selection and single-cell cloning.The infectivity efficiency was observed,expressing gene and protein levels were detected through RT-qPCR,Western Blot,and indirect immunofluorescence assays,and an EGFR protein overexpression MDCK cell line was established.Using flow cytometry,apoptosis and cell cycle of overexpression and control cell lines were separately determined.The canine EGFR gene was located on chromosome 18 and consisted of 30 exons encoding a transmembrane glycoprotein.Through the reaction system,PCR products were ligated into linearized expression vectors,generating 8 positive transformants with a size of 738 bp.The obtained lentivirus titer of EGFR gene overexpression was 3.5×10⁸ TU/mL.Transfected MDCK cells exhibited superior fluorescent effect was remarkable under fluorescent microscopy,resulting in a significant increase in the expression level of the EGFR gene and its protein within the cells.Indirect immunofluorescence assay revealed a substantial enhancement in the expression of EGFR in cells.In contrast,early apoptosis and late apoptosis rates among the transfected cells increased notably,with a clear arrest occurring at the G2/M phase.This research successfully developed a stable canine EGFR overexpressing MDCK cell line.Overexpression of canine EGFR stimulated cell division,elevated DNA replication,and concurrently triggered programmed cell death.

The aim of this study was to investigate the effect of Ezrin on the maturation of mouse oocytes,fertilization,and early embryo development,as well as to reveal its mechanisms.Mouse germinal vesicle(GV)stage oocytes and pronuclear-stage embryos were injected with siRNA targeting Ezrin(si-Ez)or a negative control siRNA(si-NC),then the rates of oocyte maturation,fertilization,and early embryo development were calculated,the length and density of microvilli on the surface of the MⅡ stage oocytes were detected with scanning electron microscopy,the spindle localization and cortical granule migration were observed using immunofluorescence staining.The results showed that knocking down Ezrin during the GV stage significantly reduced the maturation rate of mouse oocytes and extremely significantly lowered the developmental rate of early-stage embryos after fertilization.While it decreased the fertilization rate of mature oocytes,the difference was not significant.Knocking down Ezrin during the pronuclear stage extremely significantly reduced the total cell number of mouse blastocysts.Although there was a downward trend in the rates of morula and blastocyst survival,there was no significant difference compared to the control group.Scanning electron microscopy revealed that knocking down Ezrin in GV stage mouse oocytes significantly decreased the length and density of microvilli on the surface of MⅡ stage oocytes.Immunofluorescence results showed that knocking down Ezrin in GV stage mouse oocytes affected the migration of the spindle and cortical granules.These results suggest that Ezrin,through its influence on microvilli formation,spindle localization,and cortical granule migration,blocks the maturation of mouse oocytes,consequently affecting fertilization and early embryo development.

In order to explore the expression of uncoupling protein 3(UCP3)in different tissues of Min pig and its effect on the expression of mitochondrial-related genes and ATP synthase-related genes in Min pig preadipocytes,the back adipose tissue of 1-month-old Min pig was collected as the research material.The preadipocytes were isolated by enzymatic digestion and cultured in vitro.At the same time,axillary fat,chest fat,back fat,inguinal fat,perirenal fat and intramuscular fat were collected to construct tissue expression profiles.Real-time Quantitative PCR was used to detect the expression of MCU family genes(MCU,MICU1,MICU2),ATP synthase(ATP5B,ATP5E)and 1,4,5-trisphosphate inositol receptor type 1(ITPR1)genes and the expression of UCP3 gene in different adipose tissues by in vitro transfection of UCP3 gene overexpression vector or interference fragment.The results showed that the enzymatic digestion method could quickly obtain sufficient preadipocytes in the back adipose tissue.The cell edge had good refractive index,clear boundary,and similar morphology to fibroblasts.UCP3 gene was expressed in different adipose tissues,with the highest expression in dorsal adipose tissue and the lowest expression in abdominal fat.After overexpression of UCP3 gene,the expression levels of MCU family genes and ITPR1 genes were significantly increased,and the expression level of ATP5B gene was significantly decreased.After UCP3 gene interference,the expression levels of MCU family genes and ITPR1 genes were significantly decreased,and the expression levels of ATP synthase genes were significantly increased.The results showed that UCP3 gene was differentially expressed in different adipose tissues,which could promote the expression of MCU family genes and ITPR1 genes and inhibit the expression of ATP5B gene.

In order to investigate the sequence characteristics of bovine Bta-miR-494 and its tissue expression,bioinformatics methods were used to analyze the characteristics of CpG islands,promoters and transcription factors of its upstream sequence and their conservatism among different species;then predicted and functionally enriched the target genes of bovine Bta-miR-494;and finally detected its spatial and temporal expression characteristics among different tissues and the same tissue at different periods of time of the bovine Bta-miR-494 by qRT-PCR method.The results showed that Bta-miR-494 was highly conserved among different species and had the closest affinity to goat,with no CpG island upstream and two transcriptional start sites;the target gene enrichment analysis showed that the target genes of Bta-miR-494 were significantly enriched in the signaling pathways related to muscle growth and metabolism,such as PI3K-Akt,p53 and MAPK; the predictive target gene enrichment analysis showed that the target genes of Bta-miR-494 were significantly enriched in the signaling pathways related to muscle growth and metabolism.qRT-PCR results showed that the expression of Bta-miR-494 was highest in the liver and lowest in the testis of adult cows;and the expression of Bta-miR-494 was significantly higher in the biceps femoris of adult cows than that of newborn calves,whereas the expression of Bta-miR-494 in the cardiac muscle and longitudinal muscle of dorsal cows was significantly lower than that of newborn calves,while the expression of Bta-miR-494 in the cardiac muscle and the longissimus dorsi muscle was significantly lower than that of newborn calves.This inter-tissue differential expression suggests that Bta-miR-494 may dominate different myogenic expression patterns during the development of muscle tissues at different growth stages in cattle.

The aim of this study was to express the ORF3 protein of Avian hepatitis E virus (aHEV)and prepare its polyclonal antibodies,which can provide biomaterials for further study on the biological function of new vaccine of aHEV ORF3 protein.The ORF3 gene of CaHEV-GDSZ01 strain was amplified by RT-PCR,ligated with pET-32a(+)expression vector to construct recombinant prokaryotic expression plasmid,and transformed into BL21(DE3)competent cells to induce expression.The expression of ORF3 protein was analyzed by SDS-PAGE gel electrophoresis and immunization of rabbits to obtain rabbit anti-ORF3 polyclonal antibody.The titer of polyclonal antibody was determined by indirect ELISA,and its specificity was detected by Western Blot and IFA.The pET-32a-ORF3 recombinant prokaryotic expression plasmid was successfully constructed,and the 27 ku insoluble recombinant protein of aHEV ORF3 was successfully expressed by prokaryotic expression system.The results of Western Blot and IFA showed that the prepared polyclonal antibody could specifically recognize ORF3 protein,and the ELISA results showed that the titer of polyclonal antibody was 1∶51 200.In this study,the ORF3 protein of CaHEV-GDSZ01 strain was successfully expressed and its polyclonal antibody was prepared.

Luchuan pig myoblast determining gene 1(MyoD1)was cloned,bioinformatics analysis was performed,and its expression was analyzed in different tissues of Luchuan pigs,to explore the role of MyoD1 gene in the growth and development of skeletal muscle.Published MyoD1 gene of wild boar sequence on NCBI as the template,designed specific primer,cloned the CDS region of MyoD1,and compared the similarity with wild boar,cattle,sheep,human,mouse and rat gene sequence,constructed system tree for bioinformatics analysis by online software.Then,the relative expression of MyoD1 gene in different tissues of Luchuan pigs by RT-PCR.The CDS of MyoD1 gene in Luchuan pig was 960 bp,encoding 319 amino acids,five base mutations.The similarities of MyoD1 genes between Luchuan pig and wild boar,cattle,sheep,human,mouse and rats were 99.2%,93.0%,92.3%,90.0%,84.3% and 84.0%,respectively.Total number of MyoD1 gene atoms in Luchuan pigs was 4 680,molecular mass of the protein was 33.99 ku,molecular formula was C₁₄₅₇H₂₂₉₆N₄₄₂O₄₇₁S₁₄;the instability coefficient was 63.87,indicating a lack of stability;isoelectric point was 5.63,which belongs to the acidic protein;no transmembrane structure existed,found 35 phosphorylation sites and 1 glycosylation site;protein secondary structure was dominated by an irregular coil,which accounting for 60.69%.The MyoD1 gene in Luchuan pigs had the highest expression in the longest dorsal muscle,which was significantly higher than in other tissues,while kidney had the lowest expression.MyoD1 gene was expressed in all tissues of Luchuan pigs and mainly in the longest dorsal muscle,we speculated that MyoD1 gene might play a crucial role in the muscle growth and development of Luchuan pigs.

This study aims to obtain the MFSD4A gene sequence in Maiwa yak and elucidate its expression patterns in different tissues,analyze the mRNA expression levels of the interacting proteins of MFSD4A,and determine the subcellular localization of the MFSD4A protein,and provide a theoretical basis for further research on regulating MFSD4A in the growth and development of yak testis.RT-PCR was used to obtain the MFSD4A gene CDS sequence in Maiwa yak and perform bioinformatics analysis.RT-qPCR was used to detect the expression differences of the MFSD4A gene in 10 tissues,including heart,liver,spleen,lung,kidney,gluteal muscle,back muscle,fat,testis,and lymph,and the correlation between the interacting proteins and MFSD4A.pEGFP-MFSD4A fusion plasmid was transfected into Madin-Darby bovine kidney cells(MDBK)to investigate the subcellular localization of the MFSD4A protein.The results indicated that the entire length of the MFSD4A gene CDS region in the macaque yak was 1 530 bp,encoding 509 amino acids with a theoretical isoelectric point(PI)of 8.66.It had 12 transmembrane domains and belonged to the alkaline transmembrane protein.RT-qPCR results showed differential expression of MFSD4A mRNA in various tissues of the macaque yak,with the highest expression in the testis.It was also highly positively correlated with the expression levels of MFSD9,CBY1,INHBA,UNC93A,SVOP,and ID1 in the testis,suggesting that the MFSD4A gene might be related to the regulation of growth and development of yak testis.Fluorescence localization of the pEGFP-MFSD4A fusion vector transfected into Madin-Darby bovine kidney cells(MDBK)showed that the protein was mainly located in the nucleus.

In order to explore the expression and function of interferon-stimulated exonuclease gene 20(ISG20)in the formation of receptivity in beef cattle endometrial epithelial cells.The CDS region of the beef cattle ISG20 was obtained by RT-PCR combined with sequencing.The sequence analysis of the ISG20 and its encoded proteins was performed using bioinformatics online tools.The expression of the ISG20 and its related genes was detected by qRT-PCR in the formation of receptivity in beef cattle endometrial epithelial cells.The results showed that the length of CDS region of the ISG20 was 516 bp,which could encode a single protein consisting of 176 amino acids with no transmembrane domain and unstable basic hydrophilicity.The protein contained the DEDD(or DnaQ-like)exonuclease superfamily domain,interacted with 10 proteins,including ISG15 and MX2,and functioned mainly in the cytoplasm.In addition,the qRT-PCR results showed that compared with the control group,the expression of the ISG20 and its related ISG15 and MX2 was both significantly up-regulated in the formation of receptivity in beef cattle endometrial epithelial cells induced by progesterone(P4)-co-interferon-tau(IFN-τ).ISG20 may be involved in the formation of receptivity in beef cattle endometrial epithelial cells and play a role in biological processes such as early pregnancy.

In order to study the structure and function of DJ-1 gene in yaks,this experiment used Meiren yak adipose tissue cDNA as a template,cloned the coding sequence (CDS) of yak DJ-1 gene using RT-PCR,and performed bioinformatics analysis,including domain prediction and protein physicochemical properties prediction of the gene sequence.Then,tissue expression analysis was performed using RT-qPCR.The results showed that the CDS region of the DJ-1 gene in Meiren yak had a total length of 570 bp and encoded 189 amino acids;the prediction of the DJ-1 domain in yaks showed that there was one PfpI domain containing the Pfam in positions 29—168 of the DJ-1 amino acid sequence.By predicting structure and function of the DJ-1 protein,the results showed no transmembrane structure and no signal peptide region;molecular weight was 20.035 31 ku,total number of atoms was 2 870,theoretical isoelectric point was 6.84,instability coefficient was 28.37,and expressed as a stable protein;the fat coefficient was 101.11,with a total average hydrophilicity coefficient of -0.004.It was a hydrophilic protein with a total of 11 phosphorylation sites.Meanwhile,the results of subcellular localization prediction indicated that the DJ-1 protein of Meiren yak was mainly distributed in the cytoplasm and mitochondria.The phylogenetic tree showed that Meiren yak had the closest genetic relationship with wild yaks and European cattle,and the farthest genetic relationship with Arctic foxes and wide snouted manatees;the RT-qPCR results showed that it was expressed in the heart,liver,spleen,lungs,muscle,fat,and testicular tissues of adult Meiren yaks,with the highest expression level in the heart and muscle tissues,and the lowest expression level in the liver and testicular tissues.

The aim of this study was to analyze the regulatory effect of RXFP2 gene on horned phenotype and related SNP markers in horned and hornless Oula sheep populations,in order to provide technical support for genetic improvement and breed breeding of Oula sheep.DNA was extracted from 100 ewe blood samples of healthy adult Oula sheep in Henan County,Qinghai Province,including 50 blood samples with horns and 50 blood samples without horns,respectively,15 samples with and without horns were randomly selected for whole genome sequencing,and then all blood samples were amplified by multiple PCR to verify the SNPs of RXFP2 gene.Genome-wide detection results showed that there were strong selection signals in chromosome 10 of Oula sheep,including RXFP2 gene,and the strongest selection signals appeared in the RXFP2 gene segment.Multiple PCR tests verified the accuracy of the 4 coding SNPs screened by whole genome resequencing.Seven high frequency SNPs in the intron region of RXFP2 gene were detected in Oula sheep(29508704G>A,29509428A>C,29509766G>A,29512170G>A,2951276G>A,29514968T>A,29521377C>A).The genotypes of the 7 SNP loci were separated in hornless phenotypes.100% of homozygous genotypes appeared horned or hornless trait,89.66% of heterozygous genotypes were hornless trait and 10.34% were horned trait.The frequency of mutant alleles was less than that of wild alleles,and the population showed an unbalanced state of Hardy-Weinberg,which meant subject to high artificial selection intensity,but the content of polymorphic information was moderate,and the potential of breeding and improvement was still large.In conclusion,the present study confirmed the strong regulatory effect of RXFP2 gene on horn trait in Oula sheep,and filtered out 7 molecular markers of RXFP2 gene on horn trait in Oula sheep population.The relevant results can be used as molecular markers for genetic improvement of the hornless population of Oula sheep.

The study aims to investigate the effect and mechanism of PPARA on intramuscular fat (IMF) deposition,and to search for molecular markers related to IMF deposition in sheep.The experiment was conducted using Small-tail Han sheep (STH) and a hybrid F₁ population between Suffolk sheep and Small-tail Han sheep(STH×SFK)as the research object.By measuring their meat quality,the histological structure and fat drop distribution of longissimus dorsi muscle of two sheep populations were compared by H&E staining and Oil Red O staining;qRT-PCR and WB techniques were used to detect the expression differences of PPARA mRNA and protein levels among different populations,and analyze the correlation between PPARA gene and IMF;Sanger sequencing technology was used to detect the PPARA gene SNP site to evaluate the possibility of using it as a genetic marker related to IMF deposition in sheep.The results indicated that the shear force and water loss rate of STH were significantly higher than those of STH×SFK,but pH was extremely significantly lower than that of STH×SFK,marble score of STH was lower than that of STH×SFK population;histological staining showed that STH muscle fibers were thick and tightly arranged,with IMF concentrated in the muscle fiber gaps,and muscle fibers were thin and loose in structure in STH×SFK,and IMF was evenly and widely distributed among muscle cell and muscle fiber spaces,with a higher content.The expression levels of PPARA mRNA in the STH were significantly higher than those in the STH×SFK population,and PPARA protein expressions of the STH were extremely significantly higher than those of the STH×SFK population.Correlation analysis showed that the expression level of PPARA gene was extremely significantly negatively correlated with sheep fat droplet area ratio and pH,extremely significantly positively correlated with shear force and water loss rate,and weakly correlated with marble score.Sanger sequencing results showed that base mutations T49885C,T50007C,G50013A,G50835A,G50942A and G51154C occurred in the second intron of PPARA gene in two sheep populations,but no C49885T mutation was detected in the STH population.The analysis of SNP genetic diversity showed that the homozygosity of SNP loci in the population was greater than the heterozygosity,and they were in a medium to low degree of polymorphism in the population;except for the G50835A mutation,all other SNPs were in line with the Hardy-Weinberg equilibrium,and with a stable genetic basis.It was believed that PPARA gene had an important impact on IMF deposition in sheep,and could be used as a potential molecular genetic marker for IMF trait selection in sheep.

The aim of this study was to investigate the effect of bisphosphoglycerate mutase(BPGM)on the growth and development of chicken myogenic cells.The transcriptomics and proteomics data was jointly analyzed in the preliminary stage of the study to identify the differentially expressed genes that might affect the growth and development,and investigated their effects on the proliferation,apoptosis and expression levels of related marker genes of myogenic cells.Inosinic acid(IMP)was detected by ELISA and adenosine triphosphate(ATP)was detected by colorimetric assay,which revealed the important roles of the key candidate genes on muscle development and meat flavor of Jingyuan chickens.The results showed that a total of 139 differentially expressed genes/proteins were identified between the breast muscle and leg muscle of Jingyuan chickens,including 75 differentially expressed genes/proteins that were up-regulated in both the transcriptome and proteome,45 differentially expressed genes/proteins that were down-regulated in both the transcriptome and proteome,13 differentially expressed genes/proteins that were down-regulated in the transcriptome,and 6 differentially expressed genes/proteins that were up-regulated in the proteome and down-regulated in the transcriptome.7 differentially expressed genes were screened from the glycolysis/glycolysis pathway,which were BPGM, phosphoglycerate kinase 1 gene(PGK1),glucose-6-phosphate isomerase gene(GPI),phosphoglycerate mutase 1 gene(PGAM1),lactate dehydrogenase A gene(LDHA),lactate dehydrogenase B gene(LDHB)and phosphoglycerate isomerase 1 gene(TPI1),and screened for the differentially expressed gene BPGM in combination with KEGG pathway enrichment analysis and protein network interactions analysis.BPGM was expressed in all tissues of Jingyuan hens,the highest expression was in pectoral muscle,and it showed an up-regulation trend during the differentiation of myoblasts.Primary myogenesis cells were isolated and cultured for cell transfection.Interfering with BPGM inhibited myoblast proliferation and promoted apoptosis;it also affected genes upstream and downstream of the glycolytic pathway,and inhibited the biosynthesis of IMP and ATP at the same time.In conclusion,down-regulation of BPGM inhibited the growth and development of myoblasts,as well as the biosynthesis of IMP and ATP.