| The harm of ovine pregnancy toxemia(OPT)to sheep production is increasing with the development of large-scale house feeding sheep industry.It is more and more urgent to study the molecular mechanism of sheep pregnancy toxemi a as well as to breed OPT-resisitant sheeps.At present,the research on OPT is mostly about pathogenesis,diagnosis and treatment,but little about the transcription regulation of sheep OPT.In the previous studies,our team analyzed the genetic variationS IN DAGLA,HMGCS2,DLD and DLAT genes,as well as the correlation between FecB mutation and OPT in sheep,and analyzed the changes of blood physiological and biochemical indexes in OPT.On this basis,300 Duhu crossbred ewes under the same feeding conditions in Henan Zhonghe animal husbandry Co.,Ltd.were selected as the experimental animals.They were treated with estrus at the same time and underwent laparoscopic uterine horn insemination at the same time.Blood samples were collected to detect physiological and biochemical indexes when they grew up naturally to 130d-145d of pregnancy in the same feeding environment.Three ewes with OPT and 3 healthy pregnant ewes were selected from the experimental sheep to collect liver tissues.IncRNA and miRNA were sequenced by Illumina hiseq 4000 high-throughput transcriptome sequencing technology.Bioinformatics analysis was carried out to find the differentially expressed genes.Seven differentially expressed genes were verified by RT-PCR to certify the accuracy of differential gene expression.The purpose of this study was to reveal the molecular mechanism of opt in sheep and provide theoretical basis for OPT resistance breeding as well as control of mutton sheep.The main results are as followsCertification of pathological states of experimental sheep:on the basis of ordinary clinic dignosis,the physiological and biochemical indexes of the experimental animals were measured at the gestational age of 130-145 days.The contents of serum glucose of 3 OPT ewes were 0.87mol/l,0.72mol/l and 0.412mol/l.while the contents of serum calcium for corresponding animals were 1.93mol/l,1.71mol/l and 1.65mol/l respectively.The contents of serum aspartate aminotransferase of 3 OPT ewes were 115.36u/l,109.76u/l and 98.65u/l,while the contents of blood D-3-hydroxybutyric acid for corresponding animals were 4.63mmol/l,4.26mmol/l and 3.43mmol/l,respectively.The 4 indice above mentioned were fully in accordance with the typical characteristic of OPT ewes according the dignosis standard of Henan province.For the control group,the contents of serum glucose of 3 ewes were 2.27mol/l,2.39mol/l and 1.99mol/l,while the contents of scrum calcium for corresponding animals were 2.31mol/l,2.45mol/l and 2.19mol,respectively.The contents of serum aspartate aminotransferase of 3 healthy ewes were 73.84U/L,70.47U/L.and 62.33U/L,while the contents of contents of blood D-3-hydroxybutyric acid for corresponding animals were 0 mmol/L.0.21 mmol/L and 0.16 mmol/L.respectively.The 4 indice for the control above mentioned were fully in accordance with the typical characteristic of healthy ewes according the dignosis standard of Henan province.mRNA defferential sequencing analysis:There were 632 significantly up-regulated mRNA s(log2-fold change>1,FDR<0.05),and 1131 significantly down-regulated mRNAs(log2-fold change<-1,FDR<0.05)in the liver samples of OPT ewes compared with the control.Go enrichment analysis of differentially expressed genes showed that the enriched functions included lipid metabolism,immune response,organic hydroxyl compound metabolism,organic acid metabolism,oxidoreductase activity and monooxygenation Enzyme activity,etc,while KEGG pathway enrichment analysis showed that the enriched pathways included Staphylococcus aureus infection,hematopoietic cell lineage,retinol metabolism,steroid biosynthesis and steroid hormone biosynthesis.lncRNA defferential sequencing analysis:the clear reads obtained from the 3 samples taken from OPT ewes were 94884372,96213418 and 87958128 respectively,while the clear reads obtained from the 3 samples taken from healthy ewes were 96439702,89700654 and 86382202,respectively.Seventeen lncRNAs were significantly up-regulated(log2-fold change>1,FDR<0.05)and 11 lncRNA were significantly down regulated(log2-fold change<1,FDR<0.05)in the liver samples of OPT ewes.Go enrichment analysis of differentially expressed genes showed that the enrichment functions included blood coagulation,coagulation,hemostasis,macrophage fusion,mitochondrial pyruvate dehydrogenase complex,pyruvate dehydrogenase complex,pyruvate dehydrogenase kinase activity,etc.,while KEGG pathway enrichment analysis showed that the enrichment pathways included B cell receptor signaling pathway,HIF-1 signaling pathway,estrogen signaling pathway,etc.miRNA differential analysis:the liver tissues of 3 OPT ewes were sequenced,and 19456217,18805068 and 19247064 clear reads were obtained from the 3 sample respectively.For the three healthy pregnant ewes sequenced,17787701,21356432 and 13428526 clean reads were obtained respectively.There were 5 up-regulated miRNAs(log2-fold change>1,FDR<0.05)and 8 down regulated miRNAs(log2-fold change<1,FDR<0.05)in the liver of OPT ewes.Go function enrichment analysis and KEGG pathway enrichment analysis showed that the differentially expressed genes were enriched in histone methyltransferase activity,GTPase catalytic activity,myoblast differentiation and other aspects,while lactose biosynthesis.pyrimidine metabolism and other pathways were significantly enriched in KEGG metabolic pathway.Verification of differentially expressed genes by fluorescent quantitative PCR:hepatic samples of 3 OPT sheep and 3 healthy pregnant ewes were collected,and the expression levels of PDK1,CD81,SETD5,SUV39H2,ARHGAP19,ARMC12 and ASHIL were detected by using fluorescent quantitative PCR methods.The results showed that the results of fluorescent quantitative PCR were consistent with those of sequencing analysis,which indicated that the sequencing analysis was reliable.In conclusion,the transcriptonle sequencing method was used to analyze the hepatic gene expression differences between 3 OPT ewes in the experimental group and 3 normal healthy pregnant ewes in the control group.There were 632 significantly up-regulated mRNA(log2-fold change>1,FDR<0.05)and 1131 significantly down regulated mRNA(log2-fold change<-1,FDR<0.05)in the experimental group.Go and KEGG enrichment analysis of differentially expressed genes showed that lipid metabolism and immune response were significantly enriched,and retinol metabolism and steroid biosynthesis were enriched.In the experimental group,17 lncRNAs were significantly up-regulated(log2-fold change>1,FDR<0.05),and 11 lncRNAs were significantly down regulated(log2-fold change<1,FDR<0.05).The differential expression genes showed that the enriched functions were mitochondrial pyruvate dehydrogenase complex,pyruvate dehydrogenase complex,pyruvate dehydrogenase kinase activity,etc.,while KEGG pathway enrichment analysis showed that the enriched pathways were B cell receptor signaling pathway There were 5 miRNAs significantly up-regulated and 8 miRNAs significantly down regulated in the liver of sheep with opt.Most of the differentially expressed genes were enriched in histone methyltransferase activity and GTPase catalyst activity,and were enriched in lactose biosynthesis and pyrimidine metabolism.The present study may be the first study on OPT at the transcriptome level,and can provide valuable information for revealing the molecular pathological mechanism and breeding for OPT resistance.Further study is needed to explain the deffrential genes. |
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