Effects Of Heat Stress On MiRNA And MRNA In Mammary Gland Of Holstein Dairy Cows

In order to investigate the regulatory role of microRNAs and RNA in mammary glands of dairy cows under heat and non-heat stress,some important microRNAs and RNA were screened for subsequent improvement of heat stress in dairy cows to improve milk yield.In this study,high throughput sequencing was used to identify the expression profiles and differential expressions of microRNAs and mRNAs in Holstein dairy cattle mammary glands during heat stress(HS)and non-heat stress(NS)periods.GO enrichment analysis and KEGG pathway analysis were carried out in order to explore the regulatory functions of related microRNAs and mRNAs in heat stress of dairy cattle.The experiment was divided into two parts:Experiment 1:The effect of heat stress on breast microRNAs in Holstein dairy cows.This part analyzed the changes of breast microRNAs in Holstein cows during heat stress and non-heat stress.Using high throughput sequencing technology,1273 microRNAs were detected in the two libraries,891 of which were expressed in both groups,315 were specifically expressed in HS group,and 67 were specifically expressed in NS group.Differential analysis revealed 138 differentially expressed microRNAs(P<0.05),95 microRNAs were up-regulated in HS group,43 microRNAs were down-regulated in HS group,of which 26 were over 100.The target genes of all microRNAs were predicted and 13140 target genes were obtained.GO enrichment and KEGG pathway analysis of 138 differentially expressed target genes of microRNAs revealed that the main metabolic pathways involved physiological processes such as MAPK signaling pathway,Endocytosis,Lysosome,apoptosis and regulation of autophagy.Further analysis showed that bta-let-7c,bta-let-7e,bta-mir-181d,bta-mir-452 and bta-mir-31,which were elevated in heat stress,might play a role in the mammary gland of heat-stressed dairy cows through the target gene IL-1.In addition,bta-mir-25 and bta-mir-382 may participate in the regulation of MAPK pathway through target gene JNK,thus affecting the function of mammary glands in dairy cows under heat stress.In summary,we found that heat stress can cause differences in the expression of specific microRNAs in dairy cows.These microRNAs participate in the corresponding metabolic pathways through target genes,thus affecting the function of mammary glands in dairy cows under heat stress.Experiment 2:Study on the effect of heat stress on mammary gland mRNA of Holstein dairy cows.The purpose of this part is to investigate the effect of heat stress on mammary gland mRNA in Holstein dairy cows.Similarly,high-throughput transcriptome sequencing was carried out in Holstein cows during heat stress and non-heat stress.The differential expression mRNA in mammary glands of cows during heat stress and non-heat stress were identified.Statistical analysis,GO enrichment analysis and KEGG pathway analysis were carried out to explore potential organisms of target genes.Learning function.Real-time fluorescence quantitative analysis of eight mRNAs was carried out,and the sequencing results were compared to verify the accuracy of RNA-seq.Specific research contents and experimental results are as follows:(1)In the mammary transcriptome sequencing of dairy cows under heat stress and non-heat stress,659 mRNAs were differentially expressed between the two groups,292 mRNAs were up-regulated during heat stress and 367 mRNAs were down-regulated during heat stress.(2)A total of 278 GO entries and 31 pathways,including Focal adhesion,platelet activation and Thyroid hormone synthesis,are significantly enriched in the differentially expressed mRNAs.Thermal stress significantly increased the expression of NR4A1,IgFBp3 and other mRNAs,which may ultimately affect the mammary gland function of dairy cows under heat stress through aldosterone synthesis,secretion and autophagy.Considering comprehensively,it can be found that heat stress can change the synthesis of related hormones in the mammary gland of dairy cows,and affect many physiological processes,eventually leading to changes in mammary gland function under heat stress.This study found that mRNAs that are differentially expressed in heat-stressed Holstein cows,(including FGFR)are predicted to be differentially expressed miRNA target genes,and that differential mRNA-enriched pathways have 10 and differential miRNAs.The enriched pathways of the target genes coincide.In addition,subtypes including miRNA's target genes predicted to be differentially expressed,such as IL-1,were detected in the form of differentially expressed genes in this study.