| The mammary gland is a special gland with lactation function,and has experienced many stages of growth,differentiation and degeneration during the growth and development of mammals.The level of milk production directly determines the mortality and growth rate of lambs during the process of sheep breeding.The genetic are particularly important factors of all factors that affect mammary gland development and lactation.Functional genes have been proven to have clear functions in the regulation of mammary gland.However,there have been no reports on the regulation of non-coding RNA in ovine mammary gland development and lactation,and its mechanism still unclear.In this study,the mammary gland tissues of threeyear-old and fourth parity small-tailed Han ewes(n=9)and Gansu alpine Merino ewes(n=9)were investigated,and transcriptome sequencing Technology(RNA-Seq),dual luciferase assay,cell culture,and real-time quantitative PCR(RT-q PCR)methods was used to explore the expression characteristics and regulatory mechanisms of non-coding RNA in sheep mammary gland development and lactation to clarify the mechanisms of sheep lactation.The study is also to provide more information for elucidating the gene regulation mechanism of sheep lactation traits and the application of molecular breeding.The main findings are as follows:1.The lactation performance of sheep is positively correlated with the size of mammary alveolar cavity and the number of organelles in mammary epithelial cells(MECs).At the peak of lactation,the alveolar cavity of small-tailed Han ewes was larger than that of Gansu alpine Merino sheep(P<0.01),and the number of rough endoplasmic reticulum and mitochondria in alveolar of ovine MECs was also more than that of Gansu alpine Merino ewes.2.The results of small RNA-Seq showed that a total of 144 known mi RNAs and 18 differentially expressed mi RNAs were found in sheep mammary glands.Among them,10 mi RNAs were up-regulated in the mammary gland tissues of Small-Tailed Han ewe during the peak lactation period,while 8 mi RNAs were significantly down-regulated in the mammary gland tissues of Small Tail Han ewes compared to Gansu alpine Merino ewes(P<0.05).GO and KEGG analysis results show that the target genes of differentially expressed mi RNAs are mainly involved in signal transduction pathways related to mammary development and lactation,such as PI3K-Akt,HIF-1 and apoptosis signal pathway.Further studies showed that mi R-432 and mi R-200 c affect proliferation and triglycerides synthesis in ovine MECs.Overexpression of mi R-432 and mi R-200 c inhibited the vitality and number of MECs,while silencing mi R-432 and mi R-200 c promoted the vitality and number of sheep MECs.The dual luciferase results showed that mi R-432 targets SCD and LPL genes.Overexpression of mi R-432 reduces the m RNA and protein expression of SCD and LPL,while silencing mi R-432 promotes the m RNA and protein expression of SCD and LPL.Overexpression of mi R-432 inhibited triglycerides synthesis and the expression of milk fat synthesis-related genes FABP4,ACACA and LPIN1,while silencing mi R-432 promoted triglyceride synthesis.mi R-200 c targets the PANK3 gene.Overexpression of mi R-200 c reduces PANK3 m RNA and protein expression,while silencing mi R-200 c promotes PANK3 m RNA and protein expression.Overexpression of mi R-200 c promoted triglyceride synthesis and the expression of milk fat synthesis-related genes FABP4,ACACA and LPIN1,while silencing mi R-200 c inhibited triglyceride synthesis.3.The results of lnc RNA-Seq showed that a total of 1,894 lnc RNAs and 68 differentially expressed lnc RNAs were found in sheep mammary gland.Among them,31 lnc RNAs were up-regulated in the mammary gland of Small-tailed Han ewes during the peak lactation period,while 37 lnc RNAs were significantly down-regulated in the mammary gland of Small Tail Han sheep compared to Gansu alpine Merino ewes(P<0.05).GO and KEGG analysis found that the target genes of differentially expressed lnc RNAs were enriched in the development,proliferation and mammary gland morphogenesis of MECs,Erb B signal transduction pathway and Wnt signal transduction pathway.Lnc RNA-mi RNA-m RNA analysis shown that lnc RNA participates in breast development and lactation by competitively binding mi RNAs.For example,MSTRG.32232.1 can competitively bind mi R-148 a and mi R-152.4.The results of circ RNA-Seq found that a total of 4,906 circ RNAs and 33 differentially expressed circ RNAs were found in the mammary gland tissues of small-tailed Han sheep and Gansu alpine Merino sheep.Among them,18 circ RNAs were up-regulated in the mammary gland of small-tailed Han sheep during the peak lactation period,while 15 circ RNAs were significantly down-regulated in the small-tailed Han sheep's mammary gland tissues compared to Gansu alpine Merino ewes(P<0.05).The results of GO and KEGG showed that the parental genes that differentially expressed circ RNAs were mainly enriched in heterocyclic compound binding,kinase activity,adhesion junction,TGF-? signaling pathway and MAPK signaling pathway.Circ RNA-mi RNA-m RNA analysis results shown that differentially expressed circ RNAs participate in mammary gland development and lactation by competitively binding mi RNAs.For example,circ-001091 can competitively bind mi R-432,mi R-200 b and mi R-29 s.5.The study found that MSRTG.7526.1 and mi R-200 c have a targeting relationship.Silencing MSRTG.7526.1 inhibited the proliferation of MECs and promoted the expression of mi R-200 c,thereby reducing the expression of PANK3.It shows that MSTRG.7526.1 can be used as the cavernous body of mi R-200 c to relieve the inhibitory effect of mi R-200 c on PANK3,thereby inhibiting the synthesis of triglycerides.In this study,we obtained the expression profiles of mi RNAs,lnc RNAs and circ RNAs in the mammary gland tissues of small-tailed Han ewes and Gansu alpine Merino ewes at the peak of lactation,and initially explored the functions of differentially expressed mi RNAs,lnc RNAs and circ RNAs in mammary gland development and lactation.Furthermore,it was found that mi R-432,mi R-200 c and MSTRG.7526.1 affected the proliferation and triglyceride synthesis of ovine MECs.The results reveal the comprehensive molecular mechanism of ovine mammary gland development and lactation,thereby providing a theoretical foundation for improving sheep's lactation performance. |
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