Analysis Of Differential MiRNAs In Duodenum Of E. Coli F18-sensitive And-resistant Weaned Piglets

larrhea and edema disease are two major infectious diseases which cause the death of post-weaning piglets, and are the diseases that lead to huge economic loss in the swine business. The major pathogen of these two diseases is enterotoxigenic E. coli F18(ECF18), small RNA library in duodenum of each E. coli F18-sensitive and-resistant weaned piglet in full-sib pair group was sequenced using Illumina Solexa high-throughput sequencing technology, and differential miRNAs were screened to provide the basis for increasing database information on pig miRNAs, understanding genetic basic differences in resistivity to E. coli F18between Chinese local pig breeds and exotic pig breeds, and finding new markers in resistivity to E. coli F18infection. The main experiment results were as followed:1. With V pattern secreting system and the fuction adhesion and acceptor test engineering, we definite the reaction between iarrhea and edema disease pathogenic bacteria and enterocyte post-weaning piglets, then it apply to the appraisement and verification of the colibacillosis resistivity and sensibility. If the small intestine epithelium of post-weaning piglets can be proved adhesed with expressed F18ab pilus typical strains107/86, inductive expression recombined rE.coli1534, pnirBMisL-fedF and987P, K88ac subunit recombined pnirBMisL-FasG, pnirBMisLFaeGfedF, it is decided as sensitivity pattern,all else,decided as resistivity pattern. In the E. coli F18sensitivity pattern and resistivity pattern of sutai pig family full-sibs breeded in the identical niche, with the starting point of the sensitivity pattern and resistivity pattern, according to the method in the front, we filted full-sib individual with the same newborn heavy, wean heavy and figure accordance from the same stemma as the pair experiment group, filting the different miRNA of the duodenum tissue, get the more direction and valueable.2. Using Illumina Solexa sequencing technology, sequencing was conducted on all individuals in E. coli F18-sensitive and-resistant groups, more than twenty million sequences were obtained from each individual, data of the E. coli F18-sensitive and-resistant groups were compared, and a total of58differential miRNAs were found, of which,46were increased and12were decreased, miRNA which mean value was more than10000by sequence counting (expression) included ssc-mir-143, ssc-let-7f, ssc-mir-192, ssc-mir-21, ssc-mir-215and ssc-mir-378. The analysis of all miRNA precursor sequence revealed that there were two mir-378precursors, namely ssc-mir-378-1and ssc-mir-378-2;precursor sequences of these differential miRNAs were compared in Sanger database, and their specific locations were determined in genome. Precursor sequences of14miRNAs were located within gene sequences, while those of45miRNAs located among gene sequences. According to the regulating relationship between miRNA and transcription factor sequences, the regulatory network of transcription factor to miRNA was obtained, a single miRNA precursor in the network was regulated by three transcription factors at most, and that is, its degree is3. They are all increased differential miRNAs. Functional significance analysis was conducted on the intersection genes of target genes corresponding to differential miRNAs and differential genes of expression profiles.Target genes corresponding to these differentially expressed miRNAs mainly involved in cell adhesion, positive regulation of transcription, positive regulation of apoptosis and response to lipopolysaccharide.3. Combined with biological functions which might be related to E. coli F18infection, further screening was conducted on the results of functional significance analysis on all target genes corresponding to increased and decreased differential miRNAs,53significant GO were obtained to construct miRNA-GO-Network with differential miRNAs, main functions of key differential miRNAs and target genes regulated by these miRNAs were selected from the samples of the two groups. Meanwhile, we used the graph theory method, the regulation status of differential miRNAs and GO in the network and key differential miRNAs and GO were obtained.using the regulatory relationship between miRNAs and target genes, miRNA-Gene-Network was constructed, we will focus on the following12miRNA, including11increased differential miRNA:ssc-mir-143, ssc-let-7f, ssc-mir-30e, ssc-mir-148a, ssc-mir-148b, ssc-mir-181a, ssc-mir-192, ssc-mir-27b, ssc-mir-15b, ssc-mir-21, ssc-mir-215, and a reduced miRNA:ssc-mir-152.4. Because no commercial TaqMan miRNA Assays quantitative Kit corresponded to ssc-mir-30e, ssc-mir-148b and ssc-mir-181a, we conducted fluorescent quantitative validation on the remaining nine target miRNAs between E coli F18-sensitive and-resistant groups with expanded sample size, and quantitative results were consistent with high-throughput sequencing results, miR-215and miR-192were significantly different between E coli F18-sensitive and-resistant groups (P<0.01), miR-143-5p and let-7f between the E coli F18-sensitive group and-resistant group (P<0.05).5. with the result of the expression chip match to the miRNA chip,we obtain significant deviation expression(P<0.05)miRNA and mRNA, there were five target genes corresponding to miR-215(ALCAM, DLG5, FRMD4B, MIPOL1, ZFHX3), there were five target genes corresponding to mir-15b, let-7f (EGLN2, CDC14B, MAP4K3, ABL2, PRKAB2), We validated the corresponded relationship between miRNA and seed sequence,then used GO and KEGG analysis to proved that all the target gene participate in cell cycle, cytoskeleton, insulin singaling and MAPK singaling.