Study On Secondary Fiber Development And Gene Expression Of Fetal Biceps Femoris In Inner Mongolia Cashmere Goats

Inner Mongolia Cashmere goat is a kind of cashmere-meat dual-purpose breed.The growth of skeletal muscle and meat quality are the key factors affecting the economic benefits of cashmere goat breeding.As the basic unit of skeletal muscle,the key period of muscle fiber formation is the late embryonic period,and the number,size and type of muscle fiber are closely related to the physical and chemical properties and sensory quality of muscle.Therefore,the fetal period is crucial for skeletal muscle development in mammals.In order to study the histological structure and variation of muscle fibers in fetal period,the number,diameter and area of biceps femoris muscle fibers in 65 d,75 d,85 d,95 d,105 d,115 d,125 d and 135 d of fetal period of cashmere goats were analyzed by HE staining.Secondly,the types of biceps femoris muscle fibers at different developmental stages were identified by immunohistochemistry.In order to further explore the related genes affecting the growth and development and quantitative proliferation of secondary muscle fibers,transcriptome sequencing was performed on the key time nodes of secondary muscle fiber development at 75 d,85 d and 95 d.The main results are as follows :1.The formation time of primary muscle fibers of Inner Mongolia cashmere goats was before 65 days.2.The primary muscle fibers and secondary muscle fibers were fused at 65 d-75 d,and the secondary fibers began to develop at 75 d.3.95 d is the turning point of goat fetal skeletal muscle fiber from proliferation and differentiation to enlargement and thickening.The development of skeletal muscle in65-95 d fetus was mainly manifested in the increase of muscle fiber number,while the development of skeletal muscle in 95 d fetus was mainly manifested in the increase of muscle fiber diameter.4.The early development of muscle fiber type in Inner Mongolia Cashmere goat is mainly slow muscle(type I muscle fiber),and the late development is mainly fast muscle(type II muscle fiber).5.Using the method of transcriptome sequencing,the average amount of data measured in each sample was 7.5 G.Differential expression analysis showed that there were 76 differentially expressed genes(P<0.05)at 75 d and 85 d,of which 45 were up-regulated and 31 were down-regulated at 75 d;there were 457 differentially expressed genes at 75 d and 95 d(P<0.05),of which 257 were up-regulated and 200 were down-regulated at 75 d.There were 171 differentially expressed genes at 85 d and95 d(P<0.05),of which 77 were up-regulated and 94 were down-regulated at 85 d.There were significant differences in gene expression profiles of E75 VSE85 VSE95 by heatmap analysis.GO functional analysis showed that the differential genes were significantly enriched in cell composition,molecular function and biological process(P<0.05).KEGG pathway analysis of differentially expressed genes enriched 276 pathways,including 22 pathways related to muscle fiber development.6.Through heatmap analysis,it was found that there were significant differences in gene expression profiles at 75 d,85 d and 95 d.Through GO function analysis,it was found that the total differential genes were enriched into 3222 GO terms,of which 2360(28.0%)differential genes were significantly enriched in Cellular Component(P<0.05),1942(23.0%)differential genes were significantly enriched in Molecular Function(P<0.05),and 4136(49.0%)differential genes were involved in Biological Process(P<0.05).KEGG pathway analysis showed that the differentially expressed genes were enriched in 276 pathways,including 22 pathways related to muscle fiber development.7.The gene regulatory networks related to secondary muscle fiber development at75 d,85 d and 95 d were constructed.The results showed that ALDOA,CAV1,CALM1,PYGM,PPP1R3 A,COL15A1,P4HA1,ENO3,CLEC3 B,TCAP and MYL1 were at the center of the regulatory network.8.Real-time fluorescence quantitative PCR(q RT-PCR)detection of candidate genes MYL1,MYLPF,ACTN3 and TNNC2 showed an upward trend from 75 d to 95 d in the fetal period of cashmere goats,which was the same as the RNA-seq results.The accuracy of RNA-seq results was verified,and the possible roles of these candidate genes in the growth and development of secondary muscle fibers and quantitative proliferation of cashmere goats were preliminarily discussed.9.The late embryonic period is a critical period for the growth of muscle fibers.Almost all muscle fibers are formed in the fetal period.There is no net increase in the number of muscle fibers after birth,and the growth ability of muscle fibers in the fetal period or determines their lifelong meat production performance.It is of great significance to study the molecular mechanism of the growth and development of secondary muscle fibers in the fetal period.