Identification Of Candidate Genes On Growth Traits Intergrating Transcriptome In Embryonic Pigsin Longissimus Muscle Tissue

Tibetan pig is a precious local pig breed mainly produced in the Qinghai-Tibet Plateau of China.The growth performance of pigs belongs to quantitative traits,which is the key field of research at present.In order to understand the regulation mechanism of muscle growth and development of Tibetan pigs,the longissimus dorsi tissues of Tibetan pigs and Yorkshire pigs at different growth stages under the same breeding method were selected as the research objects.RNA-Seq technology,RT-q PCR technology,SNP screening and transcription factor prediction were used to explore the regulation mechanism of pig muscle growth from the level of genomics and m RNA.Furthermore,it provides a theoretical basis for exploring the functional genes of muscle quality of Tibetan pigs and the application of molecular breeding.1.Based on the analysis of RNA-seq data,the screening criteria were set as: | log2 Fold Change|> 0,Padj<0.05.The results showed that a total of 6163 differential genes were screened in 1-day-old and30-day-old groups(TP1vs TP30),7 322 genes were screened in 1-day-old and 90-day-old groups(TP1vs TP90),and 6943 genes were screened in1-day-old and 180-day-old groups(TP1vs TP180).The differential gene(DEGs)was analyzed by GO and KEGG,and the selected DEGs was mainly concentrated in the entries and pathways related to muscle growth,such as glucagon signal pathway,ribosome,lysosome and Rap1 signal pathway.A total of 5 247 differential genes were screened in 1-day-old and 30-day-old groups(LW1vs LW30),6 823 genes in 1-day-old and90-day-old groups(LW1vs LW90),and 6 632 genes in 1-day-old and180-day-old groups(LW1vs LW180).The DEGs was analyzed by GO and KEGG,and the selected DEGs was mainly concentrated in the items and pathways related to muscle growth,such as chemokine signal pathway,c GMP-PKG signal pathway,lysosome and Rap1 signal pathway.2.180-day-old is the critical period of pig growth,which may be the key node affecting pig growth and development.The data of longissimus dorsi tissue transcriptome of 180-day-old Tibetan pigs and Yorkshire pigs were selected and analyzed.According to the screening criteria of |log2Fold Change|>0,padj<0.05.There were 3 181 differentially expressed genes in TP and LW groups,including 1 632 up-regulated genes and 1549 down-regulated genes.GO enrichment and KEGG pathway analysis of these 3 181 DEGs showed that they were enriched in growth factor binding,calcium binding,actin cytoskeleton,growth and growth regulation and other related biological functions.The enrichment pathways include p53 signal pathway,Ras signal pathway,Ma PK signal pathway,Fo XO signal pathway and glucagon signal pathway,which are related to the mechanism of muscle growth regulation.Combined with previous studies and functional annotation of differential genes,10 important candidate genes that may be related to muscle growth were screened,which are GADD45 A,FBXO32,CXCL10,GLUL,TFRC,IGFBP5,ARG2,GDNF,DDC and REV3 L genes.3.Through RT-q PCR technique,it was found that the expression of GADD45 A,GLUL,DDC and REV3 L gene in Tibetan pig was significantly higher than that in Yorkshire pig,the expression of FBXO32,CXCL10 and GDNF gene in Tibetan pig was significantly higher than that in Yorkshire pig,the expression of TFRC and ARG2 gene in Tibetan pig was significantly lower than that in Yorkshire pig,and the expression of IGFBP5 gene in Tibetan pig was significantly lower than that in Yorkshire pig.The m RNA expression levels of the 10 candidate genes were consistent with the transcriptome data,which further verified the reliability and authenticity of the transcriptome data.4.At present,there is no related literature on the polymorphism of CXCL10,FBXO32 and GADD45 A genes in Tibetan pigs and Yorkshire pigs,so we choose to conduct in-depth study on these three important candidate genes.Four mutation sites were screened from the 5 ’flanking region of CXCL10,among which two mutation sites A-808G、T-832 G may be related to muscle growth.Five mutation sites were screened from the 5 ’flanking region of FBXO32,among which two mutation sites G-159C、G-137 A may be related to muscle growth.Four mutations were screened from the 5 ’flanking region of GADD45 A,among which two mutations,G-1952 A and T-1689 C,may be related to muscle growth.At the same time,the six mutation sites of T-832G、A-808G、G-159C、G-137A、G-1952A、T-1689 C were predicted,and it was found that the type and number of transcription factors changed before and after the mutagenesis.These changes may affect gene expression abundance.To sum up,transcriptome analysis was used to study the longissimus dorsi tissue of Tibetan pigs and Yorkshire pigs,and the differentially expressed genes related to muscle growth were screened,and the important candidate genes were verified at m RNA level,SNPs sites were screened and transcriptional factors were predicted,so as to provide a theoretical basis for elucidating the molecular mechanism of muscle growth regulation in Tibetan pigs.It is of great significance to provide guidance and reference for the study of muscle growth-related genes and for an in-depth understanding of muscle growth regulatory network.