| The problem of limb and hoof is one of the main reasons that restrict the productivity of breeding pigs and lead to the elimination of sows,which causes huge economic losses to the pig industry of our country.Pig tibia plays an important role in supporting weight,and its normal development can ensure the basic health of sows’limbs and hooves,so the study of tibia development is particularly important.There are many factors affecting the foot-and-limb problem,among which genetic factors are considered to be the main factors,but the molecular genetic mechanism is not clear.The previous work of our group introduced the technique of bone mineral density(BMD)measurement to determine the related traits of limb-hoof firmness,which confirmed that the limb-hoof firmness had moderate heritability,but it was not clear whether there were differences in bone molecular genetic mechanism among different varieties.In this study,the right tibia of Duroc,Landrace and Yorkshire piglets at 1 and 28 days of age were collected for tissue section observation,blood samples were collected for analysis of bone specific markers.In addition,tissue samples of the left tibia were collected for RNA-seq sequencing and transcriptome analysis was used to identify the differentially expressed genes between the two periods to reveal the molecular mechanism affecting tibia development.The main test results are as follows:(1)The tibia slices of 1-day-old and 28-day-old were observed by staining with hematoxylin-eosin staining(HE),and the results of observation of tibia sections of three breeds of piglets were synthesized.it was found that the density of bone cortical layer of 28-day-old piglets was slightly higher than that of 1-day-old,and trabecular bone density at 28-day-old is less than 1-day-old.Therefore,this study concluded that the appearance of tibia of 28-day-old piglets was harder,but the resistance to external stress decreased.(2)the Alkaline phosphatase(ALP),a marker of bone formation,was measured and analyzed by automatic serum biochemical analyzer.The ALP concentration of Duroc piglets at the 1-day-old was 1272.822±103.809 U/L,and the concentration at the 28-day-old was 332.567±16.351 U/L.The concentration of ALP at the 28-day-old very significantly lower than 1-day-old(P<0.001);The ALP concentration of Landrace piglets at 1-day-old was 1712.000±309.902 U/L,and the concentration at 28-day-old was 415.522±116.657 U/L,the ALP concentration at 28-day-old was significantly lower than that at 1-day-old(P<0.01);The ALP concentration of Yorkshire piglets at1-day-old was 1223.478±362.268 U/L,and the concentration at 28-day-old was371.389±126.393 U/L,the concentration of ALP at 28-day-old was significantly lower than that at 1-day-old(P<0.05).The bone resorption marker C-terminal telopetide of type I collagen(CTX)was measured and analyzed.the CTX concentration at 1-day-old for Duroc piglets was 16.41±1.14 nmol/L,and the CTX concentration at 28-day-old was 15.09±2.08 nmol/L;the CTX concentration at 1-day-old for Landrace piglets was13.39±0.30 nmol/L,CTX concentration at 28-day-old was 15.45±0.73 nmol/L,CTX level at 28-day-old was significantly higher than 1-day-old(P<0.05);CTX concentration of Yorkshire piglets at 1-day-old was 17.97±1.45 nmol/L,the CTX concentration at 28-day-old was 18.31±2.47 nmol/L.According to the comprehensive analysis of the results of serum biochemical indexes,it is concluded that during the development of tibia of piglets from 1 day to 28 days old,bone development is mainly regulated by the level of bone formation,while the effect of bone resorption is small.(3)The transcriptome maps of 1-day-old and 28-day-old tibia of three breeds of piglets were compared and analyzed.555 differentially expressed genes were found in the tibia data of Duroc piglets at different ages,and these differentially expressed genes were enriched in mineral absorption,rheumatoid arthritis and c GMP-PKG signal pathway.563 differentially expressed genes were found in tibia data of Landrace piglets at different ages.It was found that these differentially expressed genes were mainly enriched in Staphylococcus aureus infection,rheumatoid arthritis,cytokine-cytokine receptor interaction and other pathways,which were similar to those of differentially expressed genes in Duroc piglets.A total of 1257 differentially expressed genes were found in the tibia data of Yorkshire piglets of different ages.Through the pathway enrichment of these differentially expressed genes,it was found that they were mainly enriched in PI3K-Akt signal pathway,PPAR signal pathway and other pathways.In terms of GO enrichment,the differential genes among the three varieties were mainly enriched in calcium homeostasis cells and response to interleukin-1.(4)By sorting the differential genes in the three varieties according to the degree of difference,the top 20 genes with the most significant difference were selected,and the genes closely related to bone development were selected as important candidate genes according to NCBI database and related literature.In this study,it was found that REG4,SLC13A2,ACTC1 and MIR133A-2 were important candidate genes affecting tibia development in Duroc piglets,IL1RAPL2,RSAD2,ISG15,ANGPTL5,HES7,MX1and CYP24A1 were important candidate genes in tibia development of Landrace piglets,and RBM46,IL1RAPL2,TRH,SDR16C5,CAV3 and CLDN3 were important candidate genes in tibia development of Yorkshire piglets.(5)In this study,through weighted gene co-expression network analysis(WGCNA),we divides genes into different modules according to expression patterns,correlation analysis was performed on the transcriptome data of 1-day-old,14-day-old and 28-day-old with period as the feature,and finds two modules with strong correlation with period.By screening Hub gene and consulting related literature,it is found that IFI44L,GHSR,CMPK2,MX1 and DDX58 have certain effects on osteogenesis,osteoclast and bone,so this study suggests that these genes play an important role in the development of tibia in three piglets.And Staphylococcus aureus infection pathway and cytokine-cytokine receptor interaction pathway are common pathways in the pathways enriched by differential genes of the three breeds,so it is considered that they play an important role in regulating the tibia development of the three breeds of piglets.(6)Among the common differential genes of the three varieties,5 genes were selected for RT-q PCR verification.The results of RT-q PCR experiment of differential genes were similar to those of transcriptome analysis,and the change trend was the same.And through the regression analysis of the difference multiple obtained by transcriptome analysis and the difference multiple obtained by RT-q PCR,we can find that R~2is about 0.8.the verification results show that the data and analysis results are reliable.To sum up,the characteristics of tibia of piglets in different periods were identified by section observation and serum biochemical index analysis.through the mining of tibia sequencing data,important candidate genes affecting tibia development of piglets were identified and their functions were annotated.at the same time,it also provides a reference for revealing the molecular mechanism of regulating tibia development of piglets,and provides a theoretical basis for improving the limb-hoof characteristics of breeding pigs. |
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