Identification And Function Of LncRNA In The Longissimus Dorsi Muscle Of MSTN-edited Meishan Pigs

Meat production traits are one of the most important economic traits of pigs.Studying the mechanism of skeletal muscles can help improve the meat production performance of pigs.The growth and development of skeletal muscle is a complex process regulated by a variety of genes and gene families,among which myostatin（MSTN）plays an important negative role.In recent years,a large number of long non-coding RNA（lncRNA）related to the growth and development of skeletal muscle has been confirmed by RNA-seq.They act as skeletal molecules,guide molecules,signal molecules and bait molecules to regulate gene expression at the chromatin,transcription and post-transcriptional levels to achieve biological functions.This study took MSTN-edited Meishan pigs as the object,combined with molecular experiments and sequencing analysis,to explore the molecular mechanism of skeletal muscle fiber growth and development.This study firstly used tissue and molecular biology methods to analyze the phenotype of MSTN-edited Meishan pig,and explored the effect of myostatin deficiency on muscle quality and type.Then,the longissimus dorsi muscle of embryonic development 65 days（E65d）was used for lncRNA sequence analysis to explore the role of lncRNA in the early development of Meishan pig skeletal muscle and the characteristics of gene expression;then,the function of these genes was analyzed in depth using bioinformatics,and lncRNA-miRNA-mRNA network was constructed,which was verified by quantitative experiments.The results are as follows:（1）The 10-month-old MSTN^-/-（double knock type）Meishan pigs could not form functional proteins due to the 11bp deletion of MSTN.Compared with MSTN^+/+（wild type）Meishan pigs,the lean meat percentage increased by about 16.53%,and the fat content decreased by about 14.18%.The increase of skeletal muscle content was mainly due to the increase of the number of muscle fibers,rather than the thickening of muscle fibers.In addition,the muscle fiber types of MSTN^-/-Meishan pigs also showed a change from slow oxidation to rapid glycolysis.（2）In this study,the lncRNA map of Meishan pig skeletal muscle development in embryonic stage was constructed,and a total of 4556 lncRNA were identified,of which 3434 were novel lncRNA,230were differentially expressed lncRNA,and 30 were significantly different.Among the predicted target genes,TNNT1 and TNNT3 were related to muscle growth and development.KEGG pathway analysis showed that MAPK pathway played a key role in the growth and development of skeletal muscle of Meishan pig strain.We also constructed a lncRNA-miRNA-mRNA network,and found a total of 8important candidate lncRNAs（LNC＿000225,XR＿001300385.2,LNC＿003357,LNC＿000140,LNC＿000893,XR＿002341394.1,XR＿002347034.1 and LNC＿000471）and 9 candidate genes（TNNT1,TNNT3,PAK1,BRAF,MEF2C,MYF5,MYH7,DAG1 and PITX3）.In addition,TUCP＿000483 may play a biological function by encoding small peptides.（3）The quantitative results of 11 randomly selected lncRNAs were consistent with sequencing,which proved the reliability of sequencing.We further verified the expression level of key LNC＿000471,and found that compared with MSTN^+/+,the expression level of LNC＿000471 in MSTN^-/-group was significantly down-regulated.And the expression level in E65d was higher than that in 4 months,8 months and 16 months.Although it was widely expressed in tissues,its expression level in skeletal muscle was higher than that in other tissues.In summary,this study found 8 important candidate lncRNAs（LNC＿000225,XR＿001300385.2,LNC＿003357,LNC＿000140,LNC＿000893,XR＿002341394.1,XR＿002347034.1 and LNC＿000471）related to the growth and development of Meishan pig skeletal muscle.These results laid a foundation for further research on the regulation mechanism of lncRNA in the development of Chinese local pig breeds Meishan pig skeletal muscle,and also provided a reference for the application of lncRNA in the genetic improvement of fat-type local pig breeds.