[ Objectivel The paper aimed to investigate the expression pattern of bbu-miR-103-1 in buffalo （Bubalus bubalis） at lactation and non-lactation periods, and to predict its target gene and function. [ Method] Express...[ Objectivel The paper aimed to investigate the expression pattern of bbu-miR-103-1 in buffalo （Bubalus bubalis） at lactation and non-lactation periods, and to predict its target gene and function. [ Method] Expression pattern of bbu-miR-103-1 at lactation and non-lactation periods were detected by qRT-PCR. The precursor expression plasmid of bbu-miR-103-1 was constructed and named LpEZX-pre-miR-103-1. It was packaged and propagated to produce high-titer lenti- virus in 293T cell lines, which could be used to infect buffalo mammary epithelial cells （BMECs） and over express bbu-miR-103-1. The inhibitor of bbu-miR- 103-1 was chemically synthesized and transfected into BMECs to suppress bbu-miR-103-1 at the same time. The relative expression of pantothenate kinase 3 （ PANK3 ） and milk fat metabolism related genes were detected by qRT-PCR. [ Result] The relative expression of bbu-miR-103-1 at lactation period was 5.29 times higher than that at non-lactation period in buffalo （ P 〈 0.01 ）. The LpEZX-pre-miR-103-1 had been successfully constructed and packaged with the infection titer of 3.47×10^6 PFU/mL. Overexpress or suppress of bbu-miR-103-1 extremely down-regulated or up-regulated the expression level of PANK3 in BMECs （ P 〈 0.01 ）. Over expression of bbu-miR-103~l extremely enhanced the expression of Acetyl-CoA carboxylase alpha（ACACA）, Glycerol-3-phosphate acyhransferase 1 mitochon- drial （GPAM）, Diacylglycerol Oacyhransferase l （DGAT1） and Pyrnvate dehydrogenase lipoamide kinase isozyme 4 （PDK4） （P 〈0.01 ）, and also significantly up-regulated the expression of sterol regulatory element binding protein-1 c （SREBPI c）, Adipose differentiation-related protein （ADFP）, Cluster of differentiation 36 （ CD36）, Acetyl-CoA synthetase short-chain subfamily member 1 （ACSS1） （P 〈0.05）. Over expression of bbu-miR-103-1 down-regulated the expression of PANK3, and improved the mRNA level of SREBPlc by feedback regulation, finally promoting the de novo synthesis of fatty acid beginning with ACACA. [ Conclusion] bbu-miR-103-1 plays an important role in enhancing milk fatty acid synthesis, which provides a molecular base for revealing formation and regulatory mechanism of high-level milk fat in buffalo.展开更多
基金Supported by National Natural Science Foundation of China(31260552)National High-tech Research and Development Plan(863 plan)(2011AA100607)+1 种基金Selection of Excellent Ecological Forage Grass Varieties and Research and Demonstration of Carbon and Nitrogen Source of Fruit-grass Coupling System(GKH 14125008-2-13)Breeding and Popularization of National Approval New Forage Variety Pennisetum purpureum(GYMK 201453057)
文摘[ Objectivel The paper aimed to investigate the expression pattern of bbu-miR-103-1 in buffalo （Bubalus bubalis） at lactation and non-lactation periods, and to predict its target gene and function. [ Method] Expression pattern of bbu-miR-103-1 at lactation and non-lactation periods were detected by qRT-PCR. The precursor expression plasmid of bbu-miR-103-1 was constructed and named LpEZX-pre-miR-103-1. It was packaged and propagated to produce high-titer lenti- virus in 293T cell lines, which could be used to infect buffalo mammary epithelial cells （BMECs） and over express bbu-miR-103-1. The inhibitor of bbu-miR- 103-1 was chemically synthesized and transfected into BMECs to suppress bbu-miR-103-1 at the same time. The relative expression of pantothenate kinase 3 （ PANK3 ） and milk fat metabolism related genes were detected by qRT-PCR. [ Result] The relative expression of bbu-miR-103-1 at lactation period was 5.29 times higher than that at non-lactation period in buffalo （ P 〈 0.01 ）. The LpEZX-pre-miR-103-1 had been successfully constructed and packaged with the infection titer of 3.47×10^6 PFU/mL. Overexpress or suppress of bbu-miR-103-1 extremely down-regulated or up-regulated the expression level of PANK3 in BMECs （ P 〈 0.01 ）. Over expression of bbu-miR-103~l extremely enhanced the expression of Acetyl-CoA carboxylase alpha（ACACA）, Glycerol-3-phosphate acyhransferase 1 mitochon- drial （GPAM）, Diacylglycerol Oacyhransferase l （DGAT1） and Pyrnvate dehydrogenase lipoamide kinase isozyme 4 （PDK4） （P 〈0.01 ）, and also significantly up-regulated the expression of sterol regulatory element binding protein-1 c （SREBPI c）, Adipose differentiation-related protein （ADFP）, Cluster of differentiation 36 （ CD36）, Acetyl-CoA synthetase short-chain subfamily member 1 （ACSS1） （P 〈0.05）. Over expression of bbu-miR-103-1 down-regulated the expression of PANK3, and improved the mRNA level of SREBPlc by feedback regulation, finally promoting the de novo synthesis of fatty acid beginning with ACACA. [ Conclusion] bbu-miR-103-1 plays an important role in enhancing milk fatty acid synthesis, which provides a molecular base for revealing formation and regulatory mechanism of high-level milk fat in buffalo.
