| With the global climate warming,the threat of high temperature to chicken breeding in summer is more and more serious.Heat stress is intensive and intensive systems affect the world’s poultry productivity and benefits of one of the main environmental stress factors,and the thermal stress is one of the most seriously affected the poultry industry environmental stressor,productivity and product quality to animal health has great negative impact on poultry temperature heat resistance between economy and have an integral role in all three have a strong negative correlation between heat stress can reduce the chicken growth speed and feed conversion rate is reduced,at the same time can make the body’s immune response is impaired,eventually led to the decrease of the chicken meat quality,can also lead to animal welfare issues.However,there are few reports on the specific mechanism of body reaction in heat stress,most of which are from the perspective of environment and nutrition.The deeper molecular mechanism still needs to be revealed through a lot of research facts.MicroRNA(miRNA)is a class of non-coding single stranded RNA molecules with a length of about 22 nucleotides,which are ubiquitous in eukaryotes and participate in the regulation of a variety of life processes.They are ubiquitous in eukaryotes and participate in the regulation of various life processes.Regulation of miRNA expression is widespread in physiological and pathological processes in organisms,and it is involved in post-transcriptional expression.This regulation is the main entry point in the field of molecular genetics.However,there are few studies on chicken heat-stress-related miRNA.Therefore,in this study,SPF chickens were selected as the experimental material,and the spleen and bursa of 3SPF chickens in the immunized group(control)and the post-immune heat stress group were taken and IIIumina was used Hi Seq2500 platform for sequencing and bioinformatics method for chicken spleen and bursa of fabricius organization miRNAs were analyzed,and through the analysis of screen differentially expressed miRNAs,at the same time,in order to study the miRNA regulation function of chicken in the process of heat stress,we use screen differentially expressed miRNA :miR-205b from the sequencing results to predict its target genes,and use the double luciferase reporter gene detection system to validate its targeted relationship.The main results are as follows:1.Through small RNA-seq,36 significantly differentially expressed miRNAs(P<0.05)were screened from the bursa of bursa after heat stress,of which 19 were significantly up-regulated and 17 were down-regulated.GO functional analysis shows that the potential target genes of candidate miRNAs were mainly enriched in nucleoside phosphorylation binding protein folding regulation of synaptic vesicle transport,endoplasmic reticulum,cardiovascular system development,vesicle mediated transport,cellular macromolecular localization,and regulation of exocytosis.KEGG pathway analysis shows that the main enrichment of potential target genes of candidate miRNAs in the endocrine system Cell cycle butyric acid metabolism endoplasmic reticulum protein processing glycerol metabolism-alanine metabolism pathway.2.Through small RNA-seq,35 significantly differentially expressed miRNAs were screened from the spleen after heat stress(P<0.05),of which 17 were significantly up-regulated and 18 were down-regulated.Go function analysis shows that the main enrichment of potential target genes of candidate miRNAs in molecular function of transcription factors combination of histone phosphatase regulator activity regulating cell metabolism regulating biological process regulating protein phosphorylation to ubiquitin protein combining immune synapse to adjust small GTP enzyme mediated signal transduction protein modification made cell metabolism process,etc.KEGG pathway analysis shows that the potential target genes of candidate miRNAs were mainly enriched in the pathway of mature oocyte PPAR ubiquitin-mediated proteolysis of mTOR 、 TGF-β which is involved in lipid metabolism.3.Bioinformatics software was used to predict the target genes of gga-miR-205b.Combined with literature reports,CD24 was selected as a candidate target gene to construct wild-type(CD24-WT)and mutant(CD24-MT)recombinant vectors for miRNA target detection.In DF-1 cells,CD24 wild-type mutant and miR-205b mimic and mimic were combined after NC co-transfection,the dual-luciferase reporter gene detection system found that the fluorescence activity of CD24-WT in the co-transfection group with miR-205b was significantly lower than that of the other three groups,indicating that miR-205b could target the 3’UTR regions of CD24 and inhibit its expression. |
