关键词: 辽宁绒山羊, 褪黑激素, FGF5, LncRNA, RNA-seq, 信号通路

Abstract:

【Objective】 The aim of this study was to screen out the LncRNA associated with villus growth in Liaoning cashmere goat skin fibroblasts, and provide basic data for the study of the function and mechanism of LncRNA related to villus growth. 【Method】 The total RNA of MT and FGF5 treated Liaoning cashmere goat skin fibroblasts was extracted, and the total RNA extracted was detected by total RNA electrophoresis detection, sequencing data quality evaluation, mapping comparison and inter-sample correlation test. The differentially expressed LncRNA was screened and its target gene was predicted. The LncRNA related to villus growth was screened by GO and KEGG enrichment analysis, and the target LncRNA was verified by Real-time PCR. 【Result】 (1) The total RNA quality of the sample showed that the RNA was in good integrity, the GC content was relatively high, the sequence was stable, and the expression level between samples was high, which met the sequencing requirements.(2) Screening of differentially expressed LncRNA showed that there were 32 differentially expressed LncRNA in 1.0g·L ^-1 24h group, 4 of which were up-regulated and 28 of which were down-regulated. There were 10 differentially expressed LncRNA in 0.2g·L ^-1 24h group, 4 of which were up-regulated and 6 were down-regulated. There were 113 differentially expressed LncRNA in the 0.2g·L ^-1 72h group, of which 5 were up-regulated and 108 were down-regulated. There were 164 differentially expressed LncRNA in the 10 ^-4g·L ^-1 24 h group, of which 70 were up-regulated and 94 were down-regulated. There were 189 differentially expressed LncRNA in the10 ^-4g·L ^-1 72 h group, of which 78 were up-regulated and 111 were down-regulated. There were 123 LncRNA differentially expressed in the 10 ^-6g·L ^-1 24 h group, among which 27 up and 96 down.(3) Target gene GO enrichment analysis showed that the 1.0g·L ^-1 24h group differentially expressed LncRNA target gene enrichment in GO's negative regulation of transcription from RNA polymerase II promoter; 0.2g·L ^-1 24h group did not differentially express LncRNA target gene enriched GO term;0.2g·L ^-1 72h group Differentially expressed LncRNA target gene enrichment in GO's cellular metabolic process biological_process, binding molecular_function, FGF5 treatment group only10 ^-4g·L ^-1 72 h group differentially expressed LncRNA target gene enriched in cell cellular_component, cell part cellular_component, intracellular cellular_component, binding molecular_function and other six items. (4) Target gene KEGG enrichment analysis showed that the differential expression of LncRNA target gene in 1.0g·L ^-1 24h group was enriched in Steroid biosynthesis pathway; in 0.2g·L ^-1 24h group, there was no differential expression of LncRNA target gene enrichment Pathway term; 0.2g·L ^-1 72h group differentially expressed LncRNA target gene enrichment in Cell cycle, DNA replication, Steroid biosynthesis, TNF, Nod-like receptor, NF-kappa B and other signaling pathways, in which TNF and NF-kappa B signaling pathways are involved in villus growth. In FGF5-treated group, differentially expressed LncRNA targets in 10 ^-4g·L ^-1 72 h group The gene was significantly enriched into nine path termes such as Fanconi anemia pathway, Huntington's disease, Metabolic pathway, Aminoacyl-tRNA biosynthesis, among which Metabolic pathway was associated with villus growth; the differentially expressed LncRNA target gene in 10 ^-4g·L ^-1 24 h group had no significant enriched pathway term;10 ^-6g·L ^-1 24 h The differentially expressed LncRNA target genes were only enriched in the Taste transduction pathway. (5) There are two LncRNA corresponding to the target genes TNFα, TNFAIP3 (A20), NFKBIA (IkBα), NFKB2 and IL8 enriched in NF-κB and TNF signaling pathways, respectively (Gene ID): XLOC_005914; XLOC_018763; There are four LncRNA corresponding to the target genes in the Metabolic pathway, namely (Gene ID): XLOC_011424, XLOC_009522, XLOC_009063, XLOC_01115. Real-time PCR results showed that LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 were consistent with high-throughput sequencing results. 【Conclusion】 LncRNA XLOC_011424, XLOC_011157, LncRNA XLOC_005914 and XLOC_018763 may increase the density and length of cashmere by regulating NF-κB, TNF or Metabolic signaling pathways related to villus growth, and thus improve the yield and quality of cashmere in Liaoning cashmere goat.

Key words: Liaoning cashmere goat, melatonin, FGF5, LncRNA, RNA-seq, signaling pathway