[ 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
The regulation of adult neural stem cells(NSCs) is critical for lifelong neurogenesis. MicroRNAs(miRNAs) are a type of small, endogenous RNAs that regulate gene expression post-transcriptionally and influence signalin...The regulation of adult neural stem cells(NSCs) is critical for lifelong neurogenesis. MicroRNAs(miRNAs) are a type of small, endogenous RNAs that regulate gene expression post-transcriptionally and influence signaling networks responsible for several cellular processes. In this study, mi R-103-3 p was transfected into neural stem cells derived from embryonic hippocampal neural stem cells. The results showed that mi R-103-3 p suppressed neural stem cell proliferation and differentiation, and promoted apoptosis. In addition, mi R-103-3 p negatively regulated Nud E neurodevelopment protein 1-like 1(Ndel1) expression by binding to the 3′ untranslated region of Ndel1. Transduction of neural stem cells with a lentiviral vector overexpressing Ndel1 significantly increased cell proliferation and differentiation, decreased neural stem cell apoptosis, and decreased protein expression levels of Wnt3 a, β-catenin, phosphor-GSK-3β, LEF1, c-myc, c-Jun, and cyclin D1, all members of the Wnt/β-catenin signaling pathway. These findings suggest that Ndel1 is a novel mi R-103-3 p target and that mi R-103-3 p acts by suppressing neural stem cell proliferation and promoting apoptosis and differentiation. This study was approved by the Animal Ethics Committee of Nantong University, China(approval No. 20200826-003) on August 26, 2020.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.
基金supported by Graduate Scientific Research Innovation Program of Jiangsu Province of China,No.KYCX192066(to WL)Project Funded by the Priority Academic Program Development(PAPD)of Jiangsu Higher Education institutions China,No.03081023(to GHJ)。
文摘The regulation of adult neural stem cells(NSCs) is critical for lifelong neurogenesis. MicroRNAs(miRNAs) are a type of small, endogenous RNAs that regulate gene expression post-transcriptionally and influence signaling networks responsible for several cellular processes. In this study, mi R-103-3 p was transfected into neural stem cells derived from embryonic hippocampal neural stem cells. The results showed that mi R-103-3 p suppressed neural stem cell proliferation and differentiation, and promoted apoptosis. In addition, mi R-103-3 p negatively regulated Nud E neurodevelopment protein 1-like 1(Ndel1) expression by binding to the 3′ untranslated region of Ndel1. Transduction of neural stem cells with a lentiviral vector overexpressing Ndel1 significantly increased cell proliferation and differentiation, decreased neural stem cell apoptosis, and decreased protein expression levels of Wnt3 a, β-catenin, phosphor-GSK-3β, LEF1, c-myc, c-Jun, and cyclin D1, all members of the Wnt/β-catenin signaling pathway. These findings suggest that Ndel1 is a novel mi R-103-3 p target and that mi R-103-3 p acts by suppressing neural stem cell proliferation and promoting apoptosis and differentiation. This study was approved by the Animal Ethics Committee of Nantong University, China(approval No. 20200826-003) on August 26, 2020.
文摘环氧基是一个非常活跃的基团,它能与酶、蛋白质和核酸等生物分子发生反应形成共价键,有利于生物分子的固定化。经共价结合法固定化的酶其稳定性及重复使用性可得到显著提高。用环氧树脂ES-103B为载体采用共价结合法对海洋细菌Bacillus sp.DL-2的胞外蛋白酶进行固定化,经过单因素实验优化条件得出最优固定化条件为:p H 8.0的胞外蛋白酶溶液,25 g/L的ES-103B,45℃下反应8h。采用此最优条件下的固定化酶拆分(±)-乙酸苏合香酯制备出了e.e.p=97.5%的(R)-1-苯乙醇(产率为45.0%)和e.e.s=99.2%的(S)-乙酸苏合香酯(产率为83.9%)。该固定化酶拆分(±)-乙酸苏合香酯在重复使用8次后制备出的(R)-1-苯乙醇的e.e.p仍大于90%,且固定化胞外蛋白酶在4℃下具有较好的储存稳定性。
