Molecular Mechanism Of The ZC3H10 Gene Promotes Adaptation To Cold Stress In Cattle

Temperature stress is one of the most important factors affecting the production performance and reproductive traits of dairy cows.In the present study,the candidate genes related to bovine cold adaptation were screened by genomic selection signal methods,the genetic variation and transcriptional regulation of candidate genes in cattle were analyzed,and the molecular mechanism of candidate genes regulating bovine cold adaptation was discussed.The results will provide a reference for the study on the regulation mechanism of cold adaptation in cattle.1.With the help of bovine high-density 777k SNP chip,the genomic selection signals related to temperature adaptation of northern and southern cattle breeds were compared and analyzed by FLK and hap FLK methods.The results showed that the ZC3H10 gene was strongly positively selected.It is suggested that ZC3H10 is involved in the regulation of cold adaptation in cattle.2.Holstein cattle were used as a model to explore the genetic variation and transcriptional regulation mechanism of ZC3H10.PCR sequencing and double luciferase report showed that,there are two SNPs in the 5’-regulatory region of ZC3H10 gene（g.-4098 G>A,g.-3409 C>A）,and their haplotypes have no significant effect on promoter activity（P>0.05）,suggesting that there are variety differences in genetic variation,and it is necessary to further analyze the genetic variation and characteristics of other local cattle populations.3.The expression level of ZC3H10 in bovine tissues was detected by RT-PCR.It was found that ZC3H10 gene was highly expressed in bovine spleen and lung,suggesting that ZC3H10 may be involved in immune response.In order to analyze the effect of low temperature on the expression of ZC3H10,bovine fetal fibroblasts（BFF）were cultured at low temperature.The results of RT-q PCR showed that the expression of ZC3H10 in BFF cultured at 28℃for 4 hours was significantly up-regulated（P<0.05）,suggesting that ZC3H10 gene plays a role in the regulation of bovine cold adaptation.4.To explore the possible role of ZC3H10 in regulating cold adaptation,a ZC3H10knockout model was constructed on BFF by using CRISPR/cas9 gene editing technology.With low temperature treatment at 28℃for 4 hours,four groups were designed:WT（wild and untreated）,KO(ZC3H10^-/-and untreated),WT＿LT（wild and low temperature treatment）and KO＿LT(ZC3H10^-/-and low temperature treatment).The analysis of transcriptome sequencing data found that differentially expressed genes participate in immune related pathways,suggesting that ZC3H10 may regulate cold stress response through some immune related pathways.In addition,some differentially expressed genes involved in PPAR signaling pathway and related to glucose and lipid metabolism and thermogenesis regulation were found and verified by RT-q PCR.The results showed that under low temperature conditions,ZC3H10 may regulate genes related to glucose and lipid metabolism and lipid transport（PLTP,APOA1,etc.）through PPAR signaling pathway,to regulate the thermogenic capacity and the adaptability to low temperature environment of cattle.In conclusion,ZC3H10 may participate in cold stress response through immune related pathways;ZC3H10 may regulate genes related to glucose and lipid metabolism and lipid transport（PLTP,APOA1,etc.）through PPAR signaling pathway,to regulate the adaptability of cattle to low temperature environment.