| Pork is the main source of animal protein in most parts of China.With the improvement of people’s living standards,pork quality has become a hot spot for consumers.The improvement of pork quality has become an urgent problem in the field of pig raising in China.The main indicators of pork quality assessment include meat color,pH,hydraulic power,intramuscular fat and tenderness.Among them,the meat color is the most intuitive indicator,and the quality of meat color directly affects consumers’ desire to purchase.The genetic improvement of pork color traits has always been a hot spot in the field of porcine genetics and breeding.Conventional breeding methods are not ideal for the improvement of pork color traits.Therefore,screening key genes affecting pork color traits and identifying valuable molecular markers is of great significance for accelerating the genetic improvement of pork color traits and improving pork quality.In this study,high-throughput sequencing technology was used to perform genome-wide transcriptome sequencing of skeletal muscles of different flesh-colored pigs.Based on differential gene expression screening,the expression level of porcine Prox1 gene(ENSSSCG00000015584)in red muscle was significantly higher than that of white muscle(log2Fold=1.53,q-value=0.0001),is a potential candidate gene affecting pork meat quality traits.Previous studies have shown that Prox1 is an important transcription factor that affects skeletal muscle growth and development.At present,the research on pig Prox1 has not been reported at home and abroad,and it is still unclear whether it will affect the pork meat quality traits.In this study,a series of candidate genes affecting pork color traits were screened by high-throughput sequencing technology,and the biological characteristics of pork color trait candidate gene Prox1 were studied,including gene mRNA sequence characteristics,expression patterns,promoter activities,and The relationship between polymorphisms in the promoter region and polymorphisms of SNPs and pork meat quality traits was established.The results of this study laid an important theoretical foundation for the subsequent genetic improvement of pork meat quality traits.The main research contents and results of this study are as follows:1.Screening candidate genes affecting pork color traits by transcriptome sequencingIn this study,six cDNA libraries,including Bf28,Bf35,Bf36,and So128,were constructed using soleus muscle(Sol,red muscle)and biceps femoris(Bf,white muscle)of three full-family Duroc × Meishan dual sows.So135 and So136,differential gene screening using high-throughput transcriptome sequencing technology.According to the strategy of two muscles in the individual,770,810 and 476 differentially expressed genes were screened from the Bf28 vs So128,Bf35 vs So135,Bf36 vs So136 comparison groups;3 databases were mixed according to the two muscles.A total of 138 differentially expressed genes were screened by the alignment method.A total of 52 overlapping differentially expressed genes were screened by combining the two methods.On this basis,the above-mentioned differentially expressed genes were used for GO enrichment analysis and KEGG metabolic pathway analysis.Finally,114 non-redundant differentially expressed genes were mapped to QTLs related to flesh-colored traits by comparing the differentially expressed genes in the quantitative trait loci(QTL)database.2 Cloning and Expression Pattern Analysis of Porcine Prox1 GeneIn this study,7-day-old Erhualian piglets were used as experimental materials,and the skeletal muscle tissues of the legs were taken for total RNA extraction and reversed into cDNA.5’ RACE,3’ RACE primers were designed based on the predicted Porx Prox1 mRNA sequence on NCBI,and three pairs of primers for the amplified coding region were designed based on the predicted mRNA.Sequencing revealed that there were two transcripts in the Prxol gene.The first transcript was 3683 bp in length and contained a 484 bp 5’ untranslated region sequence,a 2214 bp coding sequence and a 985 bp 3’ untranslated region sequence.The second transcript is 4236 bp in length and includes a 1037 bp 5’untranslated region sequence,a 2214 bp coding sequence,and a 985 bp 3’ untranslated region sequence.Real-time PCR was used to analyze the expression patterns of Prox1 gene in different tissues of Duroc × Meishan binary hybrid adult pigs,70-day long white fetal pigs,and Landrace pigs at different developmental stages.The results of the study were significant.The Prox1 gene was highly expressed in liver and heart tissues,but was low in lung,spleen,kidney,stomach and adipose tissue.In the expression pattern of skeletal muscle,the expression level of Prox1 gene in soleus muscle(red muscle)was significantly higher than that of biceps femoris(white muscle)(P<0.01).The expression patterns of different developmental stages indicate that the expression level of Prox1 gene before birth is higher than that after birth.In addition,the expression of Prox1 and three myosin heavy chains(MyHCI,MyHCIIB,MyHCIIX)and Myoglobin was compared and analyzed using the longest muscle sample of the skin group of Pi× Du × Chang × Big Four.The results showed that the expression pattern of Prox1 gene in the high and low flesh color group was consistent with MyHCI and Myoglobin;the expression pattern of MyHCIIB was opposite.3.Identification of the core promoter region of Porcine Prox1 gene.In this study,five different fragments of two different promoter regions of the two transcripts of the Prox1 gene were inserted into the pGL3.0-Basic vector,and t he promoters of the two transcripts were respectively constructed into five double lu ciferase reporter vectors.The promoter fragment deletion vectors of the first transcri pt were named pGL3.0-Basic-P11(+122/+318),pGL3.0-Basic-P12(-192/+318),pGL3.0-Basic-,respectively.P13(-682/+318),pGL3.0-Basic-P14(-1182/+318),pGL3.0-Basi c-P15(-1957/+318);promoter fragment deletion vector of the second transcript They are named pGL3.0-Basic-P21(-146/+117),pGL3.0-Basic-P22(-458/+117),pGL3.0-Basi c-P23(-1080/+117),pGL3.0-Basic-P24(-1584/+117),pGL3.0-Basic-P25(-2178/+117).The above 10 vectors were transfected into 293T cells and PK15 cells,respectively,and finally analyzed by dual luciferase activity.Analysis of luciferase activity in di fferent fragments of the first promoter region revealed that+122/+318 had the stron gest fluorescence activity in 293T cells,while at-192/+318,-682/+318,-1182/+318 The fluorescence activity of the four fragments is gradually weakened,and negative regulatory elements may be present.The-1182/-1957 bp region has enhanced fluore scence activity,and this fragment may have positive regulatory elements.In PK15 c ells,the fragment luciferase activity differed from that of 293T cells.The two frag ments-192/+318 and-1822/+318 have the highest luciferase activity,and the corres ponding regions may have positive regulatory elements.The three luciferase activitie s of the three fragments+122/+318,-682/+318,and-1957/+318 were weak.The an alysis of the dual luciferase activity of different fragments of the second promoter r egion revealed that the fluorescence activity of the pGL3.0-Basic-P21(-146/+117)fra gment was the highest in both cells,indicating that the region was the first.The co re area of the two promoters.4.Association analysis between molecular polymorphism of pig Prox1 gene promoter region and pork quality traits.This study used a targeted sequencing method for 200 pigs including 66 scalp × Du ×Chang × Big Four pigs,22 Landrace pigs,22 Big White pigs,9 Duroc pigs,21 two-faced faces,20 heads.Meishan,20 rice pigs,and 20 kb regions of the first transcript promoter region of the Prox1 gene of 20 Suhuai pigs were targeted for sequencing.A total of 18 mutation sites were identified by sequencing,and the genetic diversity of 18 mutation sites in different pig breeds was analyzed.The analysis found that 8 of the mutation sites have extensive polymorphisms in different pig breeds.On this basis,using PCR-RFLP typing method,three SNPs,SNV11(g.-930bp),SNV14(g.-1421bp),and SNV18(g.-1421bp)were 279 scalp × du × long × large The quaternary commercial pigs were genotyped and the association between the polymorphisms of these loci and 11 phenotypic traits was performed using SAS software.The genotyping results showed that the three were in close chain.Correlation analysis showed that the polymorphism of the three sites was significantly correlated with the pH24h phenotype(P=0.022),and the correlation with the 24h drip loss was close to the significant level(P=0.07).In summary,the pig Prox1 gene is an important candidate gene affecting pork quality.These results provide a theoretical basis for the genetic improvement of pork quality traits using the Prox1 gene. |
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