| Animal meat products are an important source of protein for humans.With the improvement of people’s living standards,the demand for animal meat products has evolved from "quantity" to "quality".The excellent characteristics of high-quality local chicken breeds make it a high-quality meat.product.The embryonic muscle growth and development determines the entire muscle process.The analysis of chicken embryonic muscle development regulatory mechanisms,candidate gene screening and functional identification provide an effective reference for high-quality local chicken breed marker-assisted selection and molecular breeding improvement,and promote poultry germplasm resources The effective development and utilization of the product has theoretical value and practical significance.In this study,the 12 th day(E12),16 th day(E16),19 th day(E19),and 21 st day(E21)of the Chengkou mountain chicken(National Breed of Livestock and Poultry Genetic Resources)embryos were used as the experimental materials,and RNA-seq was used.The technology explores the law of muscle development in different time periods of embryos,selects m RNAs and lnc RNAs that are differentially expressed in time series;predicts the function of lnc RNAs through bioinformatics methods,constructs a molecular regulatory network for embryonic muscle development,and compares them at the individual and cellular levels Preliminary exploration of the function of candidate genes.The main results are as follows:(1)Using HE staining to explore the law of embryonic muscle development,the results show that Chengkou mountain chicken leg muscle fibers have a different developmental state from Roman layer and broiler.Chengkou mountain chicken leg muscle fiber has not formed a complete morphological structure at E12,while the other two kinds of chickens had formed complete muscle fiber structure at E12.The statistics of muscle fiber diameter and area of Chengkou Mountain Chicken embryos at different time points showed that the muscle fiber diameter and area increased significantly with time(P < 0.01);Chengkou Mountain Chicken muscle fiber diameter and area were significantly smaller than that of broiler and Roman layers at E21(P < 0.05),and the diameter and area of muscle fibers in broilers were significantly larger than those of Roman layers(P < 0.05).(2)12 Chengkou mountain chicken embryonic leg muscle c DNA libraries(E12-1,E12-2,E12-3,E16-1,E16-2,E16-3,E19-1,E19-2,E19-3,E21-1,E21-2 and E21-3)were constructed.(3)A total of 16,330 m RNAs were identified by m RNA sequencing using 12 c DNA libraries,including 109 newly predicted m RNAs transcripts,and 6,726 differentially expressed genes(DEGs)were screened out by comparison and analysis at different time points;analyzed by time sequence analysis The temporal and spatial expression patterns of DEGs at different time points were constructed to construct a clustering diagram of DEGs expression trends.In addition,Venn was used to screen out 50 candidate genes differentially expressed at different time points,and time series expression pattern analysis and functional annotation were carried out for the 50 candidate genes.(4)12 c DNA libraries were used for lnc RNA sequencing,and a total of 9,107 lnc RNAs transcripts were identified,including 874 new lnc RNA transcripts(using CPC and CNCI to predict the coding ability of the transcripts,and taking the intersection of the two as new lnc RNAs).A total of 1057 differentially expressed lnc RNAs were screened out through the comparison and analysis of differences at different time points.(5)Association analysis between lnc RNAs and protein-coding genes was conducted to predict lnc RNAs involved in the regulation of antisense,cis and trans,and to construct m RNA-lnc RNA gene regulation network.Functional annotation of target genes of lnc RNAs that play different biological roles was performed,among them,GO functional analysis found that a large number of items related to transcriptional regulation and muscle development were significantly enriched(P <0.05),while KEGG pathways analysis showed that a large number of molecular metabolic pathways related to muscle development were significantly enriched(P < 0.05),suggesting that lnc RNAs may participate in the biological process of muscle development by affecting the expression of these protein-coding genes.(6)The lnc RNA 7280.4 and its target gene STMN1 were screened,and the expression pattern of lnc RNA 7280.4 and STMN1 in muscle tissue was explored.Tissue expression profiles found that both were highly expressed in leg muscles and breast muscles;lnc RNA 7280.4 and STMN1 were highly expressed in Chengkou mountain chicken,Roman layer and white feather broiler E12 breast and leg muscles.With the passage of embryonic development time,lnc RNA 7280.4 and STMN1 The expression of STMN1 was significantly down-regulated(P < 0.05);the correlation analysis of the expression of lnc RNA7280.4 and STMN1 found that they are highly correlated in different tissues,further indicating that lnc RNA 7280.4 affects muscle development by regulating the expression of STMN1.(7)Preliminary exploration of cell function of lnc RNA7280.4 and its target gene STMN1.The results showed that the expression of STMN1 and lnc RNA 7280.4 changed significantly with the proliferation time(P <0.05);with the passage of myoblast differentiation time(myoblast differentiation and myotube fusion),the expression of STMN1 and lnc RNA 7280.4 gradually decreased,The expression pattern of STMN1 and lnc RNA 7280.4 is similar to that of pectoralis and leg muscle tissues at different development times.In this study,through the screening and identification of m RNAs and lnc RNAs related to muscle development in the embryonic leg muscles of Chengkou Mountain Chicken at different time points,as well as the preliminary exploration of the function of lnc RNA 7280.4/STMN1.The results provided a data basis for further exploring the molecular mechanism of muscle development of chengkou mountain chickens,and provided an effective reference for further marker-assisted selection and molecular breeding improvement of Chengkou Mountain Chicken. |
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