Screening And Identification Of LncRNA Related To Muscle Development In Dazu Black Goats

In recent years,with the improvement of people’s living standards,consumers’ demand for mutton products has also increased.However,the per unit yield level of mutton breeds in my country is low,especially the carcass weight and slaughter rate of goats are significantly lower than those of fine breeds such as Boer goats,which seriously restricts the development of my country’s meat goat breeding industry.To improve goat meat production,it is necessary to pay attention to goat muscle growth and development.Therefore,it is urgent to explore the molecular regulation mechanism of goat muscle development.As important transcriptional regulators in organisms,a large number of studies have shown that long non-coding RNA(lncRNA)can participate in various biological pathways,such as cell proliferation and differentiation,ontogeny,protein modification,and disease occurrence.And studies have shown that lncRNAs can regulate the growth and development of livestock muscles by acting on skeletal muscle satellite cells(SMSCs).In this study,RNA-Seq technology was used to compare and analyze lncRNAs in the longissimus dorsi muscle tissue of Dazu black goat at the two key stages of embryonic stage 75 d and postnatal 1d,to screen the differentially expressed lncRNAs in skeletal muscle tissue of goats at different developmental stages,and to annotate their functions.Further screening of lncRNAs and m RNAs related to muscle development will provide a scientific basis for studying the molecular regulation mechanism of skeletal muscle growth and development in Dazu black goat.The main results of this study are as follows:1.RNA sequencing data: The number of raw reads in the c DNA sequencing libraries of 12 longissimus dorsi tissue samples was ranged from 73,036,538 to159,613,839 in this study.The number of clean reads retained after quality control is72,804,930 ～ 159,280,432,and the comparison rate with the goat reference genome ARS1 is 91.62% ～ 94.37%,of which 88.06% ～ 91.31% of the clean reads have unique genomic positions.And the reads ratio of each sample meets the Q20 is 97.56% ～98.57%,which proves that the data sequencing is effective.2.Differential analysis of lncRNA and m RNA between groups: A total of 5,699 lncRNA transcripts were identified in this study,including 4,479 known lncRNAs and1,220 novel lncRNAs.The longissimus dorsi muscle tissue at postnatal 1d was used as the control group for the differential analysis between groups,and a total of 648 differentially expressed lncRNAs(DElncRNA,P < 0.05)were identified,of which 375 were up-regulated and 273 were down-regulated;a total of 4,517 differentially expressed m RNAs(DEm RNA,P < 0.05)were identified,of which 3,140 were up-regulated and 1,377 were down-regulated.3.DElncRNA target gene prediction: A total of 4,784 target genes significantly associated with lncRNAs were identified in the target relationship prediction of 648 DElncRNAs in the study.GO and KEGG enrichment analysis showed that a large number of target genes were significantly enriched in 855 GO terms(P < 0.05)and 47 KEGG pathways(P < 0.05).These included a large number of GO terms and KEGG pathways related to muscle development,indicating that lncRNA can be involved in the biological process of muscle development.4.Using q PCR to verify the accuracy of RNA-Seq results: 10 DElncRNAs and 20 DEm RNAs were randomly selected for q PCR verification.The results showed that the change trend of lncRNA and m RNA expression was consistent with the results of RNA-Seq,and the agreement rate was 90%.It shows that the RNA-Seq results have high confidence.5.Identification of SMSC in goats: The primary SMSCs were isolated and purified,and identified by immunofluorescence.The results showed that Pax7 gene labeling and DAPI staining were effective.During the differentiation process of goat SMSCs,the morphological changes of SMSCs and myotube formation could be observed.And the two differentiation marker genes of SMSC(Myo G and My HC)were stably expressed,indicating that the SMSC was in good condition and a stable cultivation system of skeletal muscle satellite cell had been established in Dazu black goat.6.LncRNA＿3390.1 regulates SMSC differentiation: The SMSCs were transfected by constructing lncRNA MSTRG.3390.1(lncRNA＿3390.1)lentiviral overexpression vector in the study.The results showed that the expression levels of cell differentiation marker genes Myo G and My HC were significantly up-regulated(P < 0.05),indicating that overexpression of lncRNA＿3390.1 can promote the differentiation of skeletal muscle satellite cells in Dazu black goat.In conclusion,a large number of lncRNAs and m RNAs related to muscle development were screened in this study,and the key candidate lncRNA＿3390.1 was identified to regulate the differentiation of skeletal muscle satellite cells in Dazu black goat.The results of this study not only provide a new understanding of the molecular genetic mechanism of goat muscle growth and development in the future,but also provide data reference for molecular genetic breeding in Dazu black goats.