| It is known to all that high-density and intensive breeding mode is easy to produce heat stress to broilers.However,the wet curtain and other improper measures to cool down the temperature is also one of the reasons for acute cold stress.The acute cold and heat stress can damage the broilers' immune function.Consequently,the acute cold and heat stress can affect the broilers' commercial production seriously and cause huge economic loss.This experiment mainly research the expressing law of the cell's immune factor gene in different immune organs of broilers under the condition of acute cold and heat stress by modern biological testing technology.This can not only reveal the regulation mechanism of miRNAs and its target gene under the condition of acute cold and heat stress,but also have great significance on providing theoretical basis for breeding new broiler breed that can resist both cold and heat condition.In this experiment,we take 45 Ross broilers which are at the age of 28 days,having similar weight and on similar health status as the research object.These broilers are divided into three groups of normal control group,cold stress group and heat stress group which stimulate the groups in the condition of acute cold stress and acute heat stress respectively in actual production.The expression quantity of eight immune cytokine genes(IL-1,IL-1?,IL-2,IL 4,IL-6,IL-12 a,IL-12?,INF-gamma)and four heat stress protein genes(Hsp60,Hsp70 and Hsp90 and Hsp110)in the spleen,the thymus,the bursa of Fabricius,liver,heart and other tissues are tested and analyzed by real-time fluorescent quantitative PCR.Furthermore,the mi RNAs in the spleen are tested by miRNA sequencing technology in order to select the mi RNAs which express differently and predict its target genes.All in all,this experiment further verifies the interactions between differentially expressed miRNAs and cytokine genes on cellular level and molecular level.Apart from that,it also reveal the regulation mechanism of differential genes and their target genes under the condition of acute cold and heat stress.All results are as follow:1.In the spleen tissue,only acute heat stress can significantly affect the expression of the IL-1? and IL-1(P<0.05).In thymus tissue,the expression of IL-12? have no significant change while the expression of other 7 cytokines declined significantly.In bursa of fabricius tissue,the IL-12? is not tested in all groups while the acute heat stress declined the expression of other cytokines genes significantly.2.In heart tissues and liver tissues,the expression of HSP70 and HSP110 have extremely significant rise under the condition of acute cold and heat stress(P<0.01).3.In spleen tissues,according to | log2 Fold | > 1 and p value < 0.05,12 differentially expressed miRNAs are selected between normal group and heat stress group.Apart from that,1 differentially expressed mi RNA is discovered between normal group and cold stress group as well as 20 differentially expressed miRNAs are selected between cold stress group and heat stress group.By predicting the target genes of all the differentially expressed miRNAs,4 target genes of the differentially expressed miRNAs are selected as the immune cytokine genes.4.Through the pathway enrichment analysis,differentially expressed miRNAs significantly enriched in the pathway of cytokines interaction under the condition of acute heat stress.5.Dual luciferase report result shows that gga-miR-34a-5p and gga-miR-449c-5p play a negative role in regulating the expression of IL-2 and IL-12?.In summary,this study demonstrated that the expression of immune gene was higher in acute heat stress than cold stress;The expression of all tested genes was the greatest in thymus tissue.Same gene had different expression law in different immune tissue.The expression of HSP70 and HSP110 have extremely significant rise under the condition of acute cold and heat stress.gga-miR-34a-5p and gga-miR-449c-5p play a negative role in regulating the expression of IL-2 and IL-12?. |
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