The Role And Mechanism Of Squalene Epoxidase(SQLE)in Lactation Of The Buffalo

The buffalo is resistant to coarse feeding,heatstress and diseasesandis,the number of buffaloes in my country ranks third in the world.With the development of buffalo milk industry in the world,improvement of milk yield has been paid more attention to indigenous buffalo in China.Compared with the induced breeds such as Mediterranean,Murra and Nili Ravi,the milk yield,milk protein percentage,milk fat percentage of indigenous buffaloes are on the low side,which seriously restricts the development of dairy buffalo industry in China.Therefore,it is urgent to improve the milk yield and quality of buffalo by molecular genetic techniques in China.In recent years,molecular breeding through marker assisted selection(MAS)and transgenic technology is atrend to improve cow's and buffalo's milk performance.The study of the regulation and mechanism of related genes on lactation is very important for the implementation of this technology.The transcriptome sequencing results of our previous study showed that,the SQLE(Squalene epoxidase)gene expression in mammary gland tissues during different lactating stages is high,so we speculate that SQLE gene was associated with milking traits of buffalo,and it may be involve in the lactation regulation function of mammary gland.This study investigated the function and mechanism of SQLE genes by both in vivo and in vitro methods to check this hypothesis.This study investigated the function and mechanism of SQLE genes by both in vivo and in vitro methods to check this hypothesis.in vivo studies,we distinguished the genetype of 5 SNPs by MALDI-TOF-MS and analysed the correlation between SNPs and 6 milking traits of the buffalo by SAS 9.4,these aimed to screen dominant genes and dominant haplotypes as molecular markers and provide technical support for the establishment of buffalo molecular marker-assisted selection.In vitro studies,we studied the SQLE gene expression in various tissues and the effect of cell proliferation,apoptosis and their related genes,the concentration of triglyceride(TG),and various casein and lipid droplet formation after SQLE over expression or interference,these aimed to further analyze the role and mechanism of SQLE genes in lactation regulation.(1)SQLE gene polymorphic lociare associated with milking traits of buffalo: Locus g14238 site was associated with milk protein percentage(P<0.05).Locus g18858 was associated with peak milk,270 d milk fat ylied and milk fat percentage significantly(P<0.05);Locus g22834 was correlated with peak milk,milk fat yield and milk protein percentage(P<0.05).The correlation between locus g22834 and milk ylied during 270 d,milk fat percentage was more obvious(P<0.01).In addition,a haplotype block containg loci g18858,g22146 and g22834 was found to associate with trait of milk fat ylied(percentage).(2)SQLE gene was associated with milking: Using various organs of buffaloes collected during the lactation period in the laboratory,we detected the expression of SQLE in mammary gland,lymph,liver,hypothalamus,stomach,large intestine,brain, pituitary,lung,spleen,ovary,uterus,fallopian tube,kidney and heart at m RNA level,the results showed that the expression of SQLE gene is highest in mammary gland.(3)SQLE gene promotes the proliferation and inhibit the apoptosis of bovine mammary gland epithelial cells(BMEC).We explored the putative function of SQLE gene though the RNA interference(RNAi)and overexpression techniques.Then we found that SQLE gene treated with RNAi could block the cell cycle at G1 phase,decrease the ability of proliferation and increase the proportion of total apoptosis in buffalo mammary epithelial cells.Moreover,the capacity of cell proliferation increase,the number of cell at the S phase increased and the proportion of total apoptosis reduced afer SQLE over expression in BMEC.The results of q RT-PCR showed that SQLE gene treated with RNAi can affect the expression of 4 growth-related genes(P21?PCNA?MYC and Bax),thereby affecting the proliferation and apoptosis of BMEC.(4)SQLE gene promotes the secretion of triglyceride and casein by BMEC: The concentration of triglyceride(TG)and ?/?-casein in the supernatant decreased(P<0.05)after SQLE gene treated with RNAi.Moreover,the concentration of triglyceride(TG)and ?/?-casein in the supernatant increased(P<0.05)and the formation of lipid droplets tended to increase after SQLE over expression in BMEC.The results of q RT-PCR showed that SQLE gene treated with RNAi could change the expression of 4 milk fat synthesis and casein metabolism-related genes(SREBP1,JAK2,e IF4 E and RPS6KB1),thereby affecting the concentration of triglyceride and casein in BMEC culture supernatant.In summary,squalene epoxidase has a regulatory effect on lactating buffalo.SQLE not only promote the proliferation and inhibit apoptosis of mammary epithelial cells,but also promote the secretion function of both milk protein and milk fat of mammary epithelial cells.Furthermore,there were 3 SNPs in the SQLE gene sequence associated with milking traits,which can be used in the establishment of molecular marker-assisted selection and transgenic breeding technology.