The Cloning Of Different Transcripts Of PRSS2 Gene And Its Regulation On Milk Lipid Metabolism In Dairy Cows

PRSS2 gene encodes serine protease 2,a trypsin family member,and takes serine as the active center to hydrolyze protein.The research on PRSS2 gene is mainly focused on pancreatitis and cancer,but our previous transcriptome analysis of our group found that the PRSS2 gene was differentially expressed in Chinese Holstein cows with low milk fat,and its genetic polymorphism was significantly correlated with meat quality and milk quality traits.These results suggest that the PRSS2 gene may play a regulatory role in the milk fat traits of dairy cows.Therefore,the PRSS2 gene was used in this experiment.Molecular biology techniques such as bioinformatics analysis,RNA interference,overexpression,RTq PCR,and Western Blot were used to explore its expression mechanism and its regulation of milk fat metabolism in bovine mammary epithelial cells to provide a molecular basis for the application of PRSS2 gene in dairy cow milk fat breeding and provide a reference for the study of other important traits and functional genes in molecular breeding.The results of this study are as follows:The c DNA of bovine mammary epithelial cells was used as the template,the CDS sequence of the PRSS2 gene was obtained by PCR technique,and the bioinformatics analysis of the PRSS2 gene was carried out.It was found that there were two transcripts of the PRSS2 gene,one with a length of 744 bp(PRSS2-1)and the other with a length of 583bp(PRSS2-2).Bioinformatics analysis showed that the minimum free energy and structural stability of the m RNA secondary structure of PRSS2-2 transcripts decreased,and the transcription appeared in advance of the stop codon,which may be translated into a small peptide.The c DNA of Chinese Simmental cattle,Leiqiong yellow cattle,and Sichuan yak were used as templates to amplify the PRSS2 gene.The results showed two transcripts of the PRSS2 gene in the above three breeds.The overexpression vectors of two transcripts of the PRSS2 gene were constructed and transfected into bovine mammary epithelial cells.The m RNA results showed that the expression level of PRSS2-2 transcripts was significantly higher than that of PRSS2-1transcripts in bovine mammary epithelial cells(P<0.05).The overexpression vectors of two transcripts of the PRSS2 gene was significantly reduced intracellular triglyceride and cholesterol,but the effect of the PRSS2-1 transcript was more significant than that of the PRSS2-2 transcript(P<0.05).The PRSS2-1 transcript of the PRSS2 gene was taken for the research.Through the construction of an over-expression and silencing vector,the involvement of PRSS2 in milk fat synthesis in bovine mammary epithelial cells was identified.The results suggested that overexpression of the PRSS2 gene in bovine mammary epithelial cells was significantly reduced triglyceride and cholesterol contents and enhanced the content of free fatty acids(P<0.05).Cross-checking bovine mammary epithelial cells transfected with the silencing of the PRSS2 gene demonstrated the silencing of the PRSS2 significantly promotes the synthesis of triglyceride and cholesterol and constrains the accumulation of free fatty acids(P<0.05).The fatty acid composition of cells was elucidated by gas chromatography-mass spectrometry(GC-MS).The results suggested that overexpression of the PRSS2 significantly lowers the contents of C17:1N7(cis-10-heptadecanoic acid)and C22:4N6(docosatetraenoic acid)(P<0.05).In contrast,the silencing of the PRSS2 significantly enhanced the contents of C17:1N7(cis-10-heptadecanoic acid)and C22:4N6(docosatetraenoic acid)(P<0.05).Taking the AMPK signal pathway as the study,the expression of lipid metabolism genes was detected through RT-q PCR and western blot.RT-q PCR results showed that overexpression of the PRSS2 gene could significantly up-regulate the expression of HSL,GSK3 B,ACSS2,SCD1,CPT1 A,Leptin,and AMPKα1 genes,while significantly downregulate the expression of SREBP1 gene(P<0.05).Interference with the PRSS2 gene could significantly down-regulate HSL,GSK3 B,ACSS2,SCD1,CPT1 A,Leptin,and AMPKα1(P<0.05)but not significantly up-regulate the m RNA expression level of the SREBP1 gene(P>0.05).The results of Western Blot further showed that overexpression of the PRSS2 gene could significantly up-regulate the expression of Leptin and AMPKα1 but significantly down-regulate the expression of SREBP1(P<0.05).The combined results of the two transcripts of the bovine PRSS2 gene,744 bp and583 bp,were found for the first time in this study.The results show that the PRSS2 gene plays an important role in regulating milk fat metabolism and may reduce triglycerides and cholesterol by regulating the Leptin/AMPKα1/SREBP1 pathway to promote the synthesis of fatty acids in bovine mammary epithelial cells.