Analysis Of Long Non-coding RNA In Hu Sheep Lamb Spleens Which Infected With Escherichia Coli F17 In Diarrhea And Non-diarrhea Group

With the improvement of consumption level,there is an increasing level in mutton popularity due to its tender,digestible and rich protein and vitamin.Escherichia coli,because of its high incidence and high mortality,has brought huge economic losses to sheep industry.There were many defects in the traditional antibiotic therapy,but starting from the genetic level could provide new ideas for improving the disease resistance of Hu sheep.In order to obtain purified Hu sheep intestinal epithelial cells,tissue block method and monoclonal method were used,and cytokeratin 18 immunofluorescence identification method was used to identify the isolated cells.RNA-seq was used to screen differential IncRNAs（DE IncRNA）from non-diarrheal and diarrheal individuals in Hu sheep after F17 E.coli infection and RT-qPCR was used to verify their expression level.GO and KEGG enrichment analysis,IncRNA-mRNA interaction analysis were used to further screen key DE IncRNAs.The expression profiles of TCONS₀0089751 were constructed in the heart,liver,spleen,lung,kidney,jejunum,ileum and duodenum of Hu sheep.Overexpression vectors of TCONS 00089751 were constructed.The expression levels of EPB41L2,FUT1/2,CDKI CDK2,CDK4 and CASP3 were detected by RT-qPCR.CCK-8 was used to detect the effect of TCONS 00089751 on the proliferation of small intestinal epithelial cells,and apoptotic test was used to detect the effect of TCONS₀0089751 on the apoptosis of small intestinal epithelial cells.Miranda was used to predict the competitive binding of microRNAs to TCONS 00089751.The targeting relationship was verified by a dual luciferase assay.The main results were as follows:1.Hu sheep intestinal epithelial cells were isolated and purified.The small intestinal epithelial cells were oval and closely connected to each other and have the same morphology.Moreover,Cytokeratin 18,the specific marker of small intestinal epithelial cells,was positively expressed by indirect immunofluorescence detection.The result indicated that Hu sheep small intestinal epithelial cells were successfully isolated and cultured in vitro,which would provided materials for subsequent gene function verification.2.34 DE IncRNAs were screened in the spleen tissues of diarrhea and non-diarrhea lambs by RNA-seq.The expression of 6 DE 1ncRNAs,which were randomly selected,showed significant differences by RT-qPCR（P<0.05）.Analysis by GO and KEGG Pathway enrichment revealed that a total of 34 IncRNAs were annotated and classified into 302 functional subclasses and 149 KEGG pathways.Through IncRNA-mRNA interaction network analysis,5 IncRNAs were screened out,and 6 co-expressed genes were found:MYO1G,TIMM29,CARM1,EPB41L2,SEPT4 and DESI2.3.The expression of TCONS 00089751 in non-diarrhea group was higher than that in diarrhea group except heart tissue,and the expression of TCONS₀0089751 in other tissues was significantly changed except liver tissue.Overexpression of TCONS 00089751 showed that the expression level of EPB41L2 changed significantly;adhesion-related gene FUT1/2 was significantly down-regulated;cell proliferation-related gene CDK1,CDK2 and CDK4 did not reach a significant level;apoptosis-related gene CASP3 was significantly up-regulated.The results of CCK-8 test showed that TCONS 00089751 had no significant effect on the proliferation of Hu sheep intestinal epithelial cells.The results of apoptosis test showed that TCONS 00089751 was beneficial to promote the apoptosis of sheep intestinal epithelial cells.9 miRNAs,which possibly competitive with TCONS 00089751,were predicted through Miranda.The targeting relationship between TCONS 00089751 and oar-miR-541-3p and oar-miR-134-3p was found to be reported by dual luciferase assays.