| The growth rate and meat quality of skeletal muscle are important economic traits in the broiler industry.The proliferation and differentiation of myoblasts are essential for the regeneration and development of skeletal muscle.Recently,more and more studies have shown that long noncoding RNAs(lnc RNAs)participate in the posttranscriptional regulation of gene expression by adsorbing mi RNAs and acting as competitive RNAs(ce RNAs).Therefore,it is of great significance to study the molecular regulation mechanism of chicken muscle development for genetic improvement of broilers.In this study,chicken primary myoblasts were isolated from Arbor Acres broiler(AA),and seven different developmental stages models were constructed.The cells were proliferated to 50%(G1)and 100%(G2),and differentiated to 1 day(D1),2 days(D2),4 days(D4),6 days(D6)and 8 days(D8)for whole transcriptome sequencing.The data were analyzed by bioinformatics to establish the expression profiles of m RNA,lnc RNA and mi RNA in chicken primary myoblasts at different developmental stages.In this study,the ce RNA regulatory network associated with lnc RNA-mi RNA-m RNA related to skeletal muscle development was deeply explored.The ce RNA regulatory network constructed by lnc CHAC1 / mi R-301a-5p / CHAC1 was used as the main line of research to reveal its role and regulatory mechanism in the proliferation and differentiation of chicken primary myoblasts and skeletal muscle development.It provides an important theoretical reference for improving chicken meat performance and meat quality in the future.The results are as follows :Part 1: The whole transcriptome sequencing analysis and ce RNA interaction network construction of chicken primary myoblastsBased on the model of different proliferation and differentiation stages of chicken primary myoblasts,6937 m RNAs,890 mi RNAs and 942 lnc RNAs were identified by whole transcriptome sequencing.In order to further lock the key genes affecting the development of chicken primary myoblasts and to deeply mine and screen the target network,by constructing a ce RNA interaction network related to skeletal muscle development,it was found that glutathione-specific γ-glutamyl transferase 1(CHAC1)is involved in the largest number of ce RNA networks.In the network associated with CHAC1,its Spearman rank correlation coefficient with mi R-301a-5p was-0.94,and the expression level of lnc RNA-MSTRG5399.1 was higher and the Pearson correlation coefficient with CHAC1 was 0.94,named lnc CHAC1.The reliability of transcriptome data was detected by q RT-PCR.In summary,this study initially constructed a potential ce RNA network involved in chicken myoblast proliferation and differentiation,lnc CHAC1/mi R-301a-5p/CHAC1,providing a reference for subsequent functional and mechanism research.Part 2: The effect of CHAC1 on the proliferation and differentiation of chicken myoblastsThe results of sequencing data and qRT-PCR showed that the expression of CHAC1 continued to increase during the differentiation of chicken primary myoblasts.Using q RT-PCR to detect the temporal expression profile and tissue expression profile of leg muscle,the results showed that CHAC1 showed a downward trend in the embryonic period of AA broiler leg muscle development,and showed a change of decreasing first and then increasing after shelling,and the expression of leg muscle and breast muscle in 1 day old was relatively high.Using gene acquisition and deletion strategies,the effects of CHAC1 on the proliferation and differentiation of myoblasts were detected by q RT-PCR,CCK-8,Ed U,flow cytometry,immunofluorescence and WB techniques.The results showed that overexpression of CHAC1 significantly promoted the proliferation and differentiation of chicken myoblasts,and the interference results were opposite.In summary,CHAC1 promotes the proliferation and differentiation of chicken myoblasts.Part 3: The identification of mi R-301a-5p target gene and its biological effect analysisThe temporal expression profile and tissue expression profile of leg muscle were detected by q RT-PCR.The results showed that the expression level of mi R-301a-5p in the temporal expression profile of leg muscle was gradually increased,and the expression level after birth was higher than that in embryonic period.The results showed that mi R-301a-5p was highly expressed in the tissue expression profile of 1 day old.The effects of overexpression and interference of mi R-301a-5p on chicken myoblasts were detected by q RT-PCR,CCK-8,Ed U,flow cytometry,immunofluorescence and WB.The results showed that mi R-301a-5p inhibited the proliferation and differentiation of chicken myoblasts.The targeting relationship was detected by double fluorescence and q RT-PCR.The results showed that mi R-301a-5p and CHAC1 could be targeted.In summary,mi R-301a-5p inhibits the proliferation and differentiation of chicken myoblasts by directly targeting CHAC1.Part 4: The effect of lnc CHAC1/mi R-301a-5p/CHAC1 on the proliferation and differentiation of chicken myoblastsThe results of qRT-PCR showed that lncCHAC1 was located in cytoplasm and nucleus.The temporal expression profile and tissue expression profile of leg muscle were detected by q RT-PCR.The results showed that lnc CHAC1 was continuously highly expressed in the embryonic period of leg muscle,and the expression level decreased sharply one day after hatching.The expression of lnc CHAC1 in 1-day-old muscle was negatively correlated with mi R-301a-5p.The effects of overexpression and interference of lnc CHAC1 on the proliferation and differentiation of chicken myoblasts were detected by q RT-PCR,CCK-8,Ed U,flow cytometry,immunofluorescence and WB.The targeting relationship was detected by double fluorescence and q RT-PCR.The results showed that mi R-301a-5p could target lnc CHAC1.The mechanism of lnc CHAC1/mi R-301a-5/CHAC1 was confirmed by complementation experiments.The results showed that lnc CHAC1 regulated the expression of CHAC1 by adsorbing mi R-301a-5p.In summary,lnc CHAC1 can act as a molecular sponge to adsorb mi R-301a-5p,thereby regulating the expression of CHAC1 and regulating the proliferation and differentiation of chicken myoblasts.The ce RNA regulatory network formed by lnc CHAC1/mi R-301a-5/CHAC1 plays an important regulatory role in the growth and development of chicken skeletal muscle.The results of this study further enriched the molecular regulatory network of skeletal muscle development and provided an important theoretical basis for the genetic improvement of chicken meat performance and meat quality in the future. |
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