Whole Transcriptome Sequencing Analysis Of Dairy Goats Injected With 5-HTP And Function Verification Of ATF3 Gene In Goat Mammary Epithelial Cells

A large amount of calcium ions were consumed by the perinatal dairy goats due to the fetal growth,the contraction of the birth canal and the formation of colostrum.Therefore,the calcium ion concentration of the blood decrease rapidly and the dairy goats are easily to occur to hypocalcemia,which leads to diseases such as paralysis,mastitis,ketosis and impregnable fetal clothing.5-HT can mobilize the release of calcium from bone to blood,and then balance the calcium homeostasis in the perinatal dairy goat.Calcium metabolism and apoptosis of mammary epithelial cells have a crucial effect on milk production performance in dairy goats,but the molecular mechanism of 5-HT that regulating calcium metabolism and apoptosis in the goat mammary epithelial cells is still unclearly.In this study,the concentration of 5-HT was increased by injecting 5-HTP in the perinatal dairy goats.Blood was collected on the day of deliver for whole transcriptome sequencing analysis.The gene which is positively regulated by 5-HT concentration and related to calcium metabolism and apoptosis was selected,then functional verification in goat mammary epithelial cells were carried,the main results obtained are as follows: 1.Whole transcriptome analysis of blood in the perinatal dairy goats which is injected with 5-HTP.In the seven days before the expected date of delivery,the concentration of 5-HT in the blood was increased by intravenous injected 5-HTP according to the weight(0.8mg/kg)every day in the dairy goats.Blood samples were collected on the day of dlivery,total RNA was extracted and library was constructed for the whole transcriptome sequencing analysis.It was screened 999 differentially expressed genes,967 differentially expressed LncRNAs,95 differentially expressed Circ RNAs and 87 differentially expressed micro RNAs.These transcripts were all regulated by the concentration of 5-HT.It was found that these differentially expressed genes and transcripts mainly affect physiological processes such as calcium metabolism,lipid metabolism,immune response,cell proliferation and cell apoptosis by functional annotation and enrichment analysis.Accordingto the mechanism of competition endogenous RNA(ce RNA),the regulatory network of LncRNA/Circ RNA-micro RNA-mRNA was constructed by Cytoscape3.6.1.It was founded that three genes in the network were related to calcium metabolism by enrichment analysis,they are TLR9,GJC2 and JPH1。The specific regulatory relationships are as follows: LncRNA MSTRG.101405.3 and MSTRG.63774.3 can competitively bind micro RNA NC＿030814.1＿19917 with JPH1 and GJC2,thereby regulating the expression of JPH1 and GJC2.LncRNA MSTRG.1354.2 can competitively bind micro RNA NC＿030825.1＿46027 with TLR9,thereby regulating the expression of TLR9.2.Influence study of ATF3 with calcium ion concentration and apoptosis in goat mammary epithelial cellsThe perinatal diary goats were injected 5-HTP,It was found 999 differentially expressed genes were regulated by 5-HT through transcriptome analysis.Combined with the transcriptome sequencing data of 5-HTP-injected mice’s mammary in the GEO database,it screened ATF3 gene was positively regulated by the concentration of 5-HT,and its function verified were carried in the goat mammary epithelial cells.The goat mammary epithelial cells were treated with 5-HTP,RT-q PCR was used to detect the expression changes of ATF3 and calcium metabolism-related genes,and fluorescent dyes were used to detect the change of intracellular calcium concentration.The results showed that 5-HTP treatment of goat mammary epithelial cells can increase the expression of ATF3(p<0.001),which is consistent with the results of transcriptome sequencing analysis in mices and dairy goats.It confirmined the accuracy of transcriptome sequencing analysis.The concentration of 5-HT in the cells was increased,the concentrateonof calcium ions in the cells was decreased(p<0.05),and the genes which is related to calcium metabolism in the cells was changed after 5-HTP treatment of goat mammary epithelial cells.It was found the SPCA1 increased(p<0.001),SPCA2 and SERCA2 decreased(p<0.001).After overexpressing/interference ATF3 in goat mammary epithelialcells,the changesof intracellular calcium ion concentration and apoptosis were detected.It was found that ATF3 reduce the intracellular calcium ion concentration andpromotet he cell apoptosis in goat mammary epithelial cells.The above research shows that the concentration of 5-HT in the blood was increased by injecting 5-HTP.The results of transcriptome sequencing analysis found that 5-HT mainly affects physiological processes such as calcium metabolism,lipid metabolism,cell proliferation,apoptosis,immune response and so on.The TLR9,GJC2 and J PH1 were found to be related to calcium metabolism in the ce RNA regulatory network.The goat mammary epithelial cells were cultured in vitro,5-HT can increase the expression of ATF3,It can reduce the intracellular calcium ion concentration and promote the cell apoptosis in the goat mammary epithelial cells.