| Hu sheep is a local sheep breed in China,which have the characteristics of early maturity,four seasons estrus,high prolificacy,good lactation performance and fast growth.It is of great significance to study the long-chain non-coding RNA(lncRNA)and genes related to the multiple fetal traits.So far,mutations in bone morphogenetic protein 1B(BMP1B/FecB),bone morphogenetic protein 15(BMPR-15/FecX)and growth differentiation factor 9(GDF9/FecG)have been found in some sheep breeds.Mutations in these genes can affect follicular development and ovulation,but the underlying genetic mechanism remains unclear.FecB is considered to be the primary gene related to sheep fecundity,and is a key candidate gene for controlling sheep reproductive performance.The results showed that FecB gene was closely related to litter size of Hu sheep,and the frequency of FecB allele was as high as 53%in Hu sheep.In recent years,;lcRNAs have attracted much attention.Studies have found that lncRNAs are involved in gonadal function,sex determination,sex hormone response,meiosis,spermatogenesis and placenta formation.In the hypothalamus-pituitary-gonad axis(HPOA),the pituitary can be regulated not only by the hypothalamus,but also by hormones and other regulatory factors,which play an important role in HPOA.When pituitary glands undergo abnormal genetic modification,they may affect the reproductive capacity of livestock through HPOA.Previous studies on reproduction mainly focused on ovarian folliculogenesis and ovulation in sheep with different fecundity,and few systematically analyzed the expression of lncRNAs in pituitary gland.Therefore,based on the FecB genotype and lambing records of Hu sheep,the pituitary gland of Hu sheep with different fecundity was screened as experimental material for sequencing analysis.It enriches the information of lncRNA in the pituitary gland of sheep and provides candidate molecules for regulating ovine prolificacy at the transcriptome level.1.Sequencing analysis of pituitary lncRNA and mRNA in high and low fecundity Hu sheepBased on FecB genotyping,the differential expression profiles of lncRNA and mRNA in pituitary gland of Hu sheep with different fecundity were analyzed.Functional clustering of COG,GO and KEGG was carried out to screen LncRNA and genes that might affect the fecundity of Hu sheep.Firstly,according to lambing records,three consecutive Hu sheep with three lambs per litter and three single lambs per litter were selected,and three of them with the same BB genotype and B+genotype were screened by FecB genotype identification.Pituitary tissues were collected after estrus synchronization.High throughput sequencing results showed that there were 298 differentially expressed genes(DE-mRNAs)and 57 differentially expressed lncRNAs(DE-lncRNAs)in pituitary tissues of high and low fecundity Hu sheep.The target genes of DE-lncRNAs and DE-mRNAs were annotated with COG function,and were classified into 17 and 11 functional categories.GO enrichment was used to screen GO items related to the reproductive capacity of Hu sheep.The top 20 GO terms with the highest enrichment degree included cell-cell adhesion,RNA-dependent DNA replication,cell respiration and spermine biosynthesis.In addition,GO terms related to reproduction are also enriched,including reproduction,reproductive process,response to stimulate and synapses.KEGG pathway enrichment analysis revealed that the pathways related to reproduction included cAMP,NF-kappa B,TGF-beta,PI3K-Akt,MAPK,hippo,cGMP-PKG and mTOR signaling pathways,as well as oxytocin,gonadotropin releasing hormone and insulin signaling pathways.Six DE-mRNAs and six DE-lncRNAs were randomly selected,the difference of expression leves was consistent with the result of sequencing.2.Interaction Analysis of lncRNA and mRNA in Pituitary of Hu Sheep with High and Low Fecundity and Study on Key GenesLncRNA are considered to be an important regulator of HPOA related to lambing performance in sheep.However,their expression patterns and potential roles in pituitary gland remain unclear.In order to further analyze and screen the potential lncRNA and mRNA that regulate the expression of genes related to Prolificacy in sheep,66 down-regulated genes and 18 up-regulated genes were constructed according to the results of COG,GO and KEGG enrichment and expression leves in experiment one.The cis-and trans-interaction maps of lncRNA with target genes were constructed by 20 lncRNA and 36 corresponding target genes.By analyzing the relationship between lncRNA-target gene-DE-mRNA,a network map of lncRNA-target gene-DE-mRNA was constructed which may affect reproduction.Two candidate lncRNAs and their four target genes SMAD2,VMAC,RANBP3 and NDUFA11 were further selected.Quantitative results showed that there was a tendency of expression in pituitary tissues of high and low fecundity Hu sheep.SMAD2 and its lncRNA MSTRG259847.2 were expressed in all tissues,and were highly expressed in pituitary tissue.By comparing the sequence of SMAD2 mRNA and protein,we constructed evolutionary tree and found that SMAD2 is conservative in different species.The expression of SMAD2 protein in high fecundity group was higher than that in low fecundity group(P<0.05).The expression of upstream genes TGF-β1,TGF-β2,TGF-β3,TGF-βR1 and TGF-βR2 in high fecundity group was higher than that in low fecundity group(P<0.05).This experiment indicated that lncRNA MSTRG.259847.2 and its predictive target gene SMAD2 may play an important role in the occurrence mechanism of high Hu fecundity sheep.3.Effects of lncRNA MSTRG.259847.2 on pituitary cells of Hu sheep in vitroThe aim of this study was to validate the target gene SMAD2 of lncRNA MSTRG.259847.2 in vitro and reveal the relationship between the target regulation of lncRNA MSTRG.259748.2 and SMAD2,and analyze its effect on reproductive hormone secretion in pituitary cells.Firstly,the pituitary cells of Hu sheep were isolated and identified,and the expression of SMAD2,a predictive target gene,was detected by interfering with lncRNA MSTRG.259847.2.The results showed that the expression of SMAD2 was decreased after lncRNA interference in Hu sheep pituitary cells(PCs).Then,the expression of hormone secretion genes in pituitary cells after interference and the changes of hormone concentration in cell culture after interference 24 were detected.The expression of MSTRG.259847.2 LH gene in the interference group was significantly lower than that in the NC group(P<0.05),and the concentration of LH hormone was also significantly lower(P<0.05).The expression of FSH gene in the interference group was lower than that in the NC group(P>0.05),but the concentration of FSH hormone was significantly lower(P<0.05).In addition,by detecting the changes of apoptosis-related genes,we found that Bcl2,caspase 3 and caspase 9 had no significant changes compared with NC group(P>0.05).Therefore,this experiment preliminarily proved that there may be a targeting regulation relationship between lncRNA MSTRG.259847.2 and SMAD2,and lncRNA MSTRG.259847.2 may affect the hormone secretion of pituitary cells by regulating the expression of SMAD2. |
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