Screening and Analysis of LncRNA Related to Muscle Development in Longissimus Dorsi Muscle of Different Sex Tan Sheep

doi:10.7668/hbnxb.20194341

Abstract

Abstract:

This study aims to analyze the effects of LncRNA on muscle development of male and female Tan sheep from the gene level,because muscle growth and development is an important indicator of economic benefit in breeding industry,and skeletal muscle development is significantly correlated with meat production performance.In order to preliminarily reveal the molecular mechanism affecting the differences in muscle development,transcriptome sequencing analysis was conducted to screen the LncRNA related to sheep muscle development.Full transcriptome sequencing (RNA-seq) and analysis were performed on longissimus dorsi muscle tissues of 3 male and female sheeps (8 months old),so as to screen out LncRNAs that may be related to muscle development of Tan sheep.The results showed that among the 8 513 LncRNAs were identified,45 intergroup differentially expressed LncRNAs were selected,of which 18 were up-regulated and 27 down-regulated.Prediction of co_location target gene of DELncRNA and GO functional enrichment analysis showed that target genes were significantly enriched in 268 GO terms,including somato-secreting cell differentiation,typical Wnt receptor signaling pathway involved in negative regulation of apoptosis,positive regulation of growth hormone secretion,and cAMP-mediated signaling.KEGG pathway enrichment analysis showed that DEGs was enriched in AMPK,NF-KB,PI3K-AKT,MAPK,cAMP,FOXO and other signaling pathways.Five LncRNAs,namely,TCONS_00017263,TCONS_00147966,TCONS_00163484,TCONS_00079802,and TCONS_00079803,which may be involved in muscle development,were further screened.Real-time Fluorescence Quantitative PCR was performed on 6 randomly selected DELs,and the expression trend was consistent with the trend of transcriptome sequencing,which further verified the accuracy of the sequencing results.These DELncRNAs obtained in the study may cause the differences in muscle growth and development of Tan sheep between males and females,and these related LncRNAs may provide more information for regulating muscle development and provide scientific basis for molecular breeding of meat performance of Tan sheep of different genders in Ningxia in the future.

Key words: Tan sheep, LncRNA, RNA-seq technology, Muscle development, qRT-PCR

SONG Yunjing, TIAN Yanmei, MENG Ke, YOU Kemei, FENG Dengzhen. Screening and Analysis of LncRNA Related to Muscle Development in Longissimus Dorsi Muscle of Different Sex Tan Sheep[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(1): 219-227. doi: 10.7668/hbnxb.20194341.

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Figures/Tables 10

Tab.1 Sequences of the primers

引物名称 Primer name	引物序列(5'—3') Primer sequence(5'—3')
TCONS_00147966	F: TGTGGAGATGCCTTTGAGAAGTG R: CTGATTGGAGCACACGGATACTG
ENSOART00000027545	F: GAGAGGGAGTGCTTGGGTGAG R: GGGCGGCTTGGTTTCTTTGG
TCONS_00199205	F: TCACAGGAATCGGAGATGAGCAG R: AGCAGTTAGGACACCACCAGAG
TCONS_00069075	F: GCCGCCGCTTCCTCTCTC R: TCAGTGACTGGACCCAGGTATTC
ENSOART00000028239	F: GCACTCACAGCAGCCAAAGG R: TCAGTCAGCACCGCATCTCC
ENSOART00000028920	F: GGGCTAGGGTGGGATGACTTC R: AACCTGGACTGGAGATTCATTTCAC
GADPH	F: TCGGAGTGAACGGATTCGG R: TGATGACGAGCTTCCCGTTC

Tab.2 Differential analysis of muscle development performance

指标 Indexes	滩羊Tan sheep
指标 Indexes	公羊(TX) Ram	母羊(T) Ewe
胴体质量/kg Carcass weight	12.77±0.41a	10.17±0.19b
活体质量/kg Live weight	26.03±1.19a	22.20±0.31b
净肉率/% Net meat percentage	33.22±3.19a	32.94±1.79a
净肉质量/kg Net meat weight	8.92±0.51a	7.18±0.30b

Tab.3 Sequencing data quality assessment

样品 Sample	原始序列 Raw reads	有效序列/% Clean reads	错误率/% Error rate	Q20 /%	Q30 /%	GC含量/% GC content	总对比数据 Total mapped	比对率/% Mapped ratio
T2X	93 427 110	92 950 110	0.03	97.86	94.08	50.97	82 633 919	88.90
T3X	81 058 112	80 854 296	0.03	97.55	93.04	48.40	72 082 560	89.15
T4X	91 217 782	90 807 628	0.02	98.04	94.26	49.15	82 525 090	90.88
T50	89 605 464	89 190 166	0.02	98.25	94.85	48.99	80 837 548	90.64
T60	86 259 834	85 850 880	0.02	98.04	94.38	49.31	77 110 013	89.82
T70	88 543 880	88 113 542	0.02	98.01	94.31	49.33	79 352 964	90.06

Fig.1 Distribution of expression levels of transcripts in each sample The box plot for each region corresponds to five statistics (maximum,upper quartile,median,lower quartile and minimum respectively from top to bottom).

Fig.2 Distribution of LncRNA type

Fig.3 Screening and statistics of DELs The left figure shows the overall distribution of differentially expressed LncRNAs,in the figure,red dots represent significantly differentially expressed up-regulated genes, and green dots represent significantly differentially expressed down-regulated genes;the diagram on the right shows the Venn diagram of the identified LncRNAs.

Fig.4 Cluster analysis of LncRNA differential expression

Fig.5 Top 30 GO terms with significant enrichment in LncRNA differential expression 1.Intracellular calcium activated chloride channel activity;2.Aspartic-type endopeptidase activity;3.Aspartic-type peptidase activity;4.5'-deoxyribose-5-phosphate lyase activity;5.Exon-exon junction complex;6.Protein localization to nuclear envelope;7.Nuclear envelope organization;8.Histone arginine methylation;9.Brush border;10.Organellar ribosome;11.Mitochondrial ribosome;12.Positive regulation of endothelial cell differentiation;13.Melanosome membrane;14.Chitosome;15.Primary alcohol metabolic process;16.DNA binding;17.Sequence-specific DNA binding;18.Basal part of cell;19.Chloride channel activity;20.Regulation of endothelial cell differentiation;21.Positive regulation of proteasomal ubiquitin-dependent protein catabolic process;22.Protein insertion into membrane;23.Dosage compensation by inactivation of X chromosome;24.Regulation of skeletal muscle cell differentiation;25.Microvillus assembly;26.Structural constituent of cytoskeleton;27.Chloride transport;28.Nuclear outer membrane;29.ATPase activator activity;30.Regulation of primary metabolic process.

Fig.6 Significantly enriched top 20 KEGG pathways with DELncRNA target genes 1.Bile secretion;2.Spliceosome;3.Primary bile acid biosynthesis;4.Insulin signaling pathway;5.Viral carcinogenesis;6.Chemical carcinogenesis;7.FOXO signaling pathway;8.Non-homologous end-joining;9.Metabolism of xenobiotics by cytochrome P450;10.Thyroid hormone signaling pathway;11.Pancreatic secretion;12.Nucleotide excision repair;13.Herpes simplex infection;14.ErbB signaling pathway;15.Fc gamma R-mediated phagocytosis;16.Pathways in cancer;17.AMPK signaling pathway;18.Protein digestion and absorption;19.Sphingolipid metabolism;20.Antigen processing and presentation.

Fig.7 qRT-PCR validation of 6 DELncRNAs

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