| China is the third largest buffalo breeding country in the world.There are27.44 million cattle in 18 southern provinces,accounting for about 20% of the total number of cattle in the country.It has become one of the important tasks of buffalo breeding to fully develop the meat performance of buffalo,further increase beef yield and improve beef quality.Muscle development mainly depends on myoblast proliferation and differentiation,which involves the precise expression and regulation of coding and non-coding genes.Micro RNA is a class of endogenous Non-coding RNA with a length of 18-23 nt.The research on the regulation of skeletal muscle has been relatively complete,but it needs to be further improved in the muscle development of buffalo.Based on high-throughput sequencing technology and bioinformatics analysis,this study identified and screened mi RNAs related to buffalo muscle development,and verified the regulatory role of bbu-mi R-493-5p,which is mainly located in the cytoplasm,on the proliferation and differentiation of buffalo myoblasts.The principal findings of the research are as follows:1.14,376,236~18,660,464 and 20,162,103~24,809,247 clean sequencing reads were acquired from a total of six sequencing libraries.at the proliferation and differentiation stages of buffalo myoblasts,and 592~620 known mi RNAs were detected at the proliferation stage.587~597 known mi RNAs were detected at the differentiation stage.59~80 new mi RNAs were detected at the proliferation stage.40~52 new mi RNAs were detected during the differentiation stage.In this sequencing,177 mi RNA were differentially expressed at the differentiation stage compared with that at the proliferation stage,88 were up-regulated and 89 were significantly down-regulated.In this sequencing,a newly discovered mi RNA with high expression in both proliferation and differentiation stages and extremely significant differential expression was named bbu-mi R-493-5p.The analysis of tissue expression profile revealed that the expression level of bbu-mi R-493-5p was significantly elevated in muscle tissue compared to other tissues.This finding signifies the potential role of bbu-mi R-493-5p in muscle biology and highlights its importance for further investigation in this tissue type;2.The effect of bbu-mi R-493-5p on the proliferation of buffalo myoblasts was detected by q PCR,CCK-8 and Ed U methods after transfecting mi RNA mimic to overexpress bbu-mi R-493-5p.The results showed that bbu-mi R-493-5p promoted the proliferation of buffalo myoblasts.The effect of bbu-mi R-493-5p on the differentiation of buffalo myoblasts was detected by q PCR,Western blotting and immunofluorescence,and the results showed that overexpression of bbu-mi R-493-5p promoted the differentiation of buffalo myoblasts.RNA-FISH analysis showed that bbu-mi R-493-5p was mainly located in the cytoplasm.In this study,through high-throughput transcriptome sequencing combined with bioinformatics analysis,mi RNAs related to muscle development of Guangxi swamp buffalo were screened out,and a newly discovered mi RNA-bbu-mi R-493-5p,which was highly expressed and significantly differentially expressed at both proliferation and differentiation stages,was selected and studied.The high expression of bbu-mi R-493-5p in muscle tissue can promote the proliferation and differentiation of buffalo myoblasts.Further,it was determined by RNA-FISH technology that the predominant site of localization for bbu-mi R-493-5p was observed within the confines of the cytoplasmic compartment.This study laid a foundation for further analysis of the regulatory mechanism of bbu-mi R-493-5p,and also provided a theoretical basis for using mi RNA as molecular markers to breed Chinese buffalo for meat. |
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