Screening And Identification Of MiRNA Differentially Expressed In Tail Fat Of Altay Sheep And Hu Sheep Under Acute Cold Stress

Object:In this study,the tail fat tissues of Altay sheep(fatty buttock tail type)and Hu sheep(less fat tail type)with obvious tail differences were used as research objects to construct mi RNA libraries of Altay sheep and Hu sheep under acute cold stress.Analysis and prediction of mi RNA target genes differentially expressed after acute cold stress and their corresponding enrichment pathways and biological functions.Screening and verifying differentially expressed mi RNAs in two libraries that may be related to fat metabolism,and laying a foundation for further research on the mechanism of mi RNAs in fat metabolism.It can provide a reference for the preliminary exploration of the molecular mechanism of sheep fat metabolism under cold stress and the breeding of new sheep breeds with higher cold resistance.Methods:This paper is divided into four experiments.The specific methods are as follows:Test 1:Collecting the tail adipose tissue of the sheep in the normal temperature group and the acute cold stress group,marked and stored them in a prepared 10% formalin solution for fixation,and then collecting the heart,liver,spleen and kidney of the sheep in the normal temperature group and the acute cold stress group.The tissues were stored in liquid nitrogen.Real-time fluorescent quantitative PCR technology was used to detected the m RNA expression of HSP60,HSP70 and HSP90 genes in the heart,liver,spleen and kidney tissues of sheep before and after acute cold stress;HE staining method was used to observe the sheep tail fat tissue under acute cold stress Morphological changes before and after.Test 2:Construction of mi RNA libraries of Altay sheep and Hu sheep under acute cold stress.The adipose tissues from the tails of Altay sheep and Hu sheep in the normal temperature group were collected in vivo,and three biological repeats each.Stored in a liquid nitrogen immediately after collection.After 24 hours of acute cold stimulation at-25 ℃,rapid bloodletting and slaughter.Collected tail fat tissue and stored in liquid nitrogen.Extracted the total RNA of 12 samples for quality inspection,and then carried out the mi RNA library construction of the samples that passed the quality inspection.The Illumina Hi Seq TM2500/Mi Seq sequencing platform was used for sequencing,and then the original data is tested for quality and high-quality data is obtained through data filtering.Test 3:Screening and identification of mi RNA differentially expressed in Altay sheep and Hu sheep,target gene prediction and bioinformatics analysis.Useing the prediction software mi REvo and mirdeep2 to predicted the new mi RNA of the matched s RNA,and analyze the matched s RNA in detail.Then screened the differentially expressed mi RNAs,and randomly selected 8-9 differentially expressed mi RNAs for q PCR to quantitatively verify their accuracy.Using mi Randa,PITA and RNAhybrid software to predicted target genes for differentially expressed mi RNAs.Useing GOseq and KOBAS software for gene enrichment analysis of target genes.Subsequently,combined with a large number of related literatures,the mi RNAs related to fat metabolism in the Altay sheep and Hu sheep libraries were screened.Test 4:Through enrichment analysis and consulting a large number of documents,a mi RNA related to fat metabolism was selected from the Altay sheep and Hu sheep libraries,respectively mi R-3957-5p andmi R-370-3p.The corresponding predicted target genes are dopamine receptor D2(DRD2)and deacetylase3(Sirtuin 3,SIRT3).Subsequently,a dual luciferase reporter gene was performed at the cellular level to detect whether mi R-3957-5p and mi R-370-3p had targeted regulatory effects on the target genes DRD2 and SIRT3.Laying the foundation for further study of mi R-3957-5p and mi R-370-3p involved in regulating fat metabolism.Result:Test 1:Observing the HE stained sections of the fat tissues of the sheep’s tail through a microscope,the morphology of adipose tissue changed before and after acute cold stress;the expression of HSPs family genes in different tissues after acute cold stress in sheep all changed significantly.This shows that the sheep model of acute cold stress was successfully constructed.Test 2:1)The total RNA quality test passed.2)Successfully constructed 6 Altay sheep mi RNA libraries and 6 Hu sheep mi RNA libraries.After sequencing,a total of 46591002 raw data were obtained from the three sample libraries(A1,A2,and A3)of Altay sheep at room temperature,a total of 52039709 original data were obtained from the three sample libraries(LA1,LA2 and LA3)of the acute cold stress group.After data filtering,a total of 46229373 data can be obtained for the follow-up experiment in the room temperature group,and the acute cold stress group obtained 51,684,440 data that can be used in subsequent experiments.The error rate of all six samples was 0.01%.A total of 45,656,439 original data were obtained from the three sample libraries(H1,H2,and H3)of the Hu sheep room temperature group.A total of47987654 raw data were obtained from the three sample libraries(LH1,LH2 and LH3)of the acute cold stress group.After data filtering,a total of 45225921 data can be used for subsequent experiments in the room temperature group,and the acute cold stress group obtained a total of 47,571,716 data that can be used for subsequent experiments.The error rate of all six samples is 0.01%.After testing,the mi RNA length of the 12 library samples is between 20～24nt,and the test meets the test requirements.Test 3:1)A total of 362 mi RNAs were detected in the Altay sheep library.There are 234 unknown mi RNAs,and the rest are known mi RNAs.Compared with the normal temperature group,25 mi RNAs were significantly differentially expressed after acute cold stress.A total of 362 mi RNAs were detected in the Hu sheep library,218 unknown mi RNAs,and the rest were known mi RNAs.Compared with the normal temperature group,24 mi RNAs were significantly differentially expressed after acute cold stress.The q PCR quantitative verification results showed that the differential expression results of the selected mi RNA were consistent with the results of high-throughput sequencing,which further proves the reliability of the data.2)GO results showed that Altay sheep group had a total of 414 enriched in biological processes,87 enriched in cellular components,and 337 enriched in molecular functions.A total of 1237 are enriched in biological processes and 236 are enriched in molecular functions in the Hu sheep group.KEGG found that the main enrichment pathways involved in candidate target genes in the Altay sheep group are Dopaminergic synapse,Linoleic acid metabolism,etc.Mi RNAs related to fat metabolism include oar-mi R-3957-5p,oar-mi R-127,oar-mi R-133,etc.The main enrichment pathways involved in candidate target genes in the Hu sheep group are the Phosphatidylinositol signaling system,the c GMP-PKG signalingpathway,etc.Mi RNAs related to fat metabolism include oar-mi R-370-3p,oar-mi R-376c-3p,oar-mi R-26 b and so on.Test 4:Double luciferase test results show that transfection of oar-mi R-3957-5p and oar-mi R-370-3p can significantly inhibit the activity of DRD2-3`-UTR-reporter gene and SIRT3-3`-UTR-reporter gene,respectively.The activity of the wild-type luciferase gene decreased,and the mutant type had no significant changes.This indicates that oar-mi R-3957-5p and oar-mi R-370-3p have targeting relationships with DRD2 and SIRT3 genes,respectively.Conclusion:(1)Successfully constructed the Small RNA library of Altay sheep and Hu sheep after acute cold stress,the library quality test was qualified,and the sheep Small RNA library was enriched.(2)Small RNA library between Altay sheep and Hu sheep is not much different.The mi RNA expression in the two libraries after acute cold stress was significantly changed,among which there were more differentially expressed mi RNAs related to fat metabolism.This shows that after acute cold stress,there will be a large number of mi RNAs involved in the regulation of fat metabolism.(3)It is proved that oar-mi R-3957-5p and oar-mi R-370-3p have targeted regulation effects on DRD2 and SIRT3,respectively.