Study On Physiology And Biochemical Reaction And It’s MRNA Transcriptional Analysis Of Leukocyte At Jersey Cow Under Summer Of Southern China

The extreme hot environment impacted and challenged the global dairy industry.Holstein is the most important dairy breed in terms of milk production.However resistance is poor,which leads to the heat stress problem of Holstein cows in summer is particularly prominent.However,Jersey cattle has good heat tolerance,so the study of Jersey cattle provides theoretical and technical support for the cultivation of excellent Jersey cattle hybrid varieties in the future.The purpose of this study was to further excavate candidate genes for heat resistance in Jersey cattle by adapting to the physiological and gene regulation mechanism of Jersey cattle in summer.In this study,Jersey cattle were used as experimental animals(n=10,340±40 kg,3.5±0.3 year).Four experiments including physiological response experiment,biochemical test,transcriptional regulation and protein expression,and were completed in Chongqing Guangda farm from July to August 2018.Respiration rate(RR),Sweating rate(SR),Rectal temperature(RT),Skin temperature with hair,(ST(hair))and Skin temperature without hair(ST(no hair))are used to evaluate the heat resistance of Jersey cattle.Blood samples were collected at different temperatures to separate serum and leukocyte.Elisa was used to detect Adenosine monophos sphate activated protein kinase(AMPK)、Heat shock transcription factor(HSF)、Leptin、Ghrelin、Na~+-K~+-ATPase.The transcriptome was sequenced by RNA-seq high-throughput sequencing technology,also related proteins were verified by Western blot,results are as follows:Results 1.The meteorological data,including dry bulb temperature,wet bulb temperature,relative humidity and temperature and humidity index(THI),were significantly affected by different time(P<0.01).The physiological indexes of thermal response of Jersey cattle,including RR、SR、ST、ST(hair)and ST(no hair),increased significantly with the increase of THI(P<0.01).Meanwhile,the serum indexes of Jersey heat response,including AMPK、HSF、Leptin、Ghrelin、Na~+-K~+-ATPase,increased significantly with the increase of THI(P<0.01).2.The transcriptome sequencing of leukocyte under three different temperature gradients was carried out.It is 170 differentially expressed genes were detected in leukocytes at 30℃,within 144 genes were up-regulated and 26 genes were down-regulated compared with 25℃.These genes were annotated into 1306 GO terms.65 differentially expressed genes were detected from leukocytes at 35℃,including 58up-regulated genes and 7 down-regulated genes,which were commented to 1147 GO term.It is 30 differentially expressed genes were detected from leukocytes at 35℃,of which 2 genes were up-regulated and 28 genes were down-regulated compared with30℃.These genes were commented to 371 GO term(FC>2,P<0.01).According to1150 heat regulatory genes and 212 differentially expressed genes,16 heat regulated differentially expressed genes were screened.3.The analysis of GO function annotation results showed that the differentially up-regulated gene of leukocyte from Jersey mainly related to 20 functions such as cellular process、single-organism process、biological regulation、cell part、organelle、membrane and binding.The analysis of KEGG functional annotation results showed that the differentially expressed genes of leukocyte from Jersey mainly focused on signaling transduction、signaling molecules and interactions、immune system、endocrine system and other functions.The fund of these functions may be related to the heat resistance of Jersey cattle.4.Through GO and KEGG enrichment analysis by transcriptome,20 and 13 signal pathways were screened by GO and KEGG,respectively.The signal pathways enriched by GO mainly include regulation of phosphorus metabolic process、response to oxygen levels、movement、positive regulation of cellular component、response to decrease oxygen levels、response to hypoxia、positive regulation of cell motility、regulation of developmental proces、cytokine activity and so on.These regulation are mainly related to oxygen regulation.KEGG-enriched signaling pathways mainly include AGE-RAGE signaling pathway in diabetes、rheumatoid arthritis、Bladder cancer、VEGF signaling pathway、Pancreatic cancer、Pathways in cancer、TNF signaling pathway、HIF-1signaling pathway and so on.VEGF can enhance the permeability of blood vessels,which is more conducive to the exchange of oxygen and other substances;under the condition of hypoxia,the extracellular matrix can be changed;the phosphorylation of the receptor itself is caused,and these functions are consistent with the results of GO analysis,therefore the research in VEGF signaling pathway is particularly meaningful.5.Correlation analysis was performed on 16 thermal regulation of differentially expressed genes,and three genes involved in VEGF signaling pathway were found.These genes were ENSBTAG00000005339,PTGS2 and ENSBTAG00000047561.6.The results of HSP70 Western blot analysis in the leukocyte of Jersey cattle were consistent with the thermally regulated differentially expressed up-regulated genes.Conclusion(1)In summer,heat-resistant molecules in serum and HSP70 in leukocytes of Jersey increased with increasing temperature.The corresponding genes such as ENSBTAG00000005339,SAMSN1,PRDM1,DNAJB13,BHLHE40 and IL1A were increased,which may be used as candidate genes for heat-resistant varieties of dairy cows.(2)Compared with 25℃,the up-regulated genes、down-regulated genes and GO term at 30℃and 35℃were 144 and 65,26 and 7,1360 and 1147,respectively.Compared with 30℃,the up-regulated genes、down-regulated genes and GO term at 35℃were 2、28 and 371,respectively(FC>2,P<0.01).According to 1550heat regulatory genes and 212 differentially expressed genes,16 heat regulatory genes were screened.(3)GO functions are concentrated in signal transduction,cellular procedures,cells and cell regions.The KEGG function focuses on functions such as signal transduction,signaling molecules and their interactions,and the immune system.(4)The results of GO and KEGG enrichment analysis are all related to oxygen regulation,so different degrees of heat stress may lead to different degrees of oxygen metabolism.(5)The thermal regulation of differentially expressed genes were screened and correlation analysis.A total of 14 genes were found to be correlated,especially ENSBTAG00000005339 was associated with the other 6 genes,and the gene was also involved in VEGF signaling pathway.Therefore,this gene may play an important role in regulating the adaptation of Jersey cattle to summer high temperature climate in southern China,and has great research value.