水牛bbu-miR-103-1在泌乳期与非泌乳期表达模式及靶向基因的初步研究  

Expression Pattern and Target Regulation Gene of bbu-miR-103-1 from Lactation and Non-lactation Periods in B.bubalis

作　　者：蔡小艳[1,2] 李胜[1] 陈秋萍[1] 王萍[1] 邓凯[1] 刘庆友[1] 石德顺[1] CAI Xiao-yan LI Sheng CHEN Qiu-ping WANG Ping DENG Kai LIU Qing-you SHI De-shun(State Key Laboratory of Tropical Biological Resources Protection and Utilization, Guangxi University, Nanning 530004, China Guangxi Institute of Animal Science, Nanning 530001, China)

机构地区：[1]广西大学亚热带农业生物资源保护与利用国家重点实验室,南宁530004 [2]广西壮族自治区畜牧研究所,南宁530001

出　　处：《畜牧兽医学报》2016年第11期2191-2201,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31260552);国家高技术研究发展计划(863计划)(2011AA100607);优势生态牧草品种遴选及果草耦合系统碳氮源汇的研究与示范(桂科合14125008-2-13);国审牧草新品种紫色象草的繁育与推广(桂渔牧科201453057)

摘　　要：旨在探讨bbu-miR-103-1在水牛泌乳期和非泌乳期的差异表达模式,并预测其靶向调控基因及功能。本研究应用qRT-PCR检测bbu-miR-103-1,在水牛泌乳期和非泌乳期的差异表达。构建bbu-miR-103-1前体表达质粒LpEZX-pre-miR-103-1,并在293T细胞中包装高滴度的慢病毒颗粒,用于感染水牛乳腺上皮细胞实现过表达bbu-miR-103-1,同时应用化学合成的bbu-miR-103-1抑制剂转染水牛上皮细胞实现抑制表达bbu-miR-103-1,研究bbu-miR-103-1对PANK3及乳脂代谢相关基因表达的影响。结果表明,水牛泌乳期bbu-miR-103-1的相对表达量为非泌乳期的5.29倍(P<0.01);成功构建的慢病毒载体LpEZX-pre-miR-103-1包装获得了感染滴度为3.47×106PFU·mL-1的慢病毒颗粒;过表达和抑制表达bbu-miR-103-1分别极显著下调和上调了水牛乳腺上皮细胞PANK3基因的相对表达量(P<0.01);过表达bbu-miR-103-1极显著提高了ACACA、GPAM、DGAT1和PDK4基因的表达(P<0.01),对SREBP1c、ADFP、CD36、ACSS1等基因产生了显著的上调作用(P<0.05)。过表达bbu-miR-103-1通过下调PANK3的表达,反馈提高了SREBP1c的mRNA水平,促进了以ACACA开头的脂肪酸从头合成。表明bbu-miR-103-1对水牛乳脂肪合成有十分重要的促进作用,为揭示水牛高乳脂形成和调控机理提供了分子依据。This study aimed to investigate the expression pattern of bbu-miR-103-1from lactation and non-lactation periods in buffalo（Bubalus bubalis）,and to predict its target gene and function.Expression pattern of bbu-miR-103-1in lactation and non-lactation periods were detected by qRT-PCR.The precursor expression plasmid of bbu-miR-103-1was constructed and named LpEZX-pre-miR-103-1（HIV）.It was packaged and propagated to produce high-titer lentivirus in293 Tcell 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 trans-fected into BMECs to suppress bbu-miR-103-1at the same time.The relative expression of pantothenate kinase 3（PANK3）and milk fat metabolism related genes were detected by qRT-PCR.The results showed that the relative expression of bbu-miR-103-1from lactation period was 5.29 times higher than that from non-lactation period in buffalo（P 〈0.01）.The LpEZX-pre-miR-103-1has been successfully constructed and packaged with the infection titer for3.47×106PFU·mL-1.Overexpress or suppress of bbu-miR-103-1extremely significantly downregulated or up-regulated（P〈0.01）the expression level of PANK3 in BMECs.Over expression of bbu-miR-103-1extremely significantly enhanced the expression of Acetyl-CoA carboxylase alpha（ACACA）,Glycerol-3-phosphate acyltransferase 1 mitochondrial（GPAM）,Diacylglycerol Oacyltransferase 1（DGAT1）and Pyruvate dehydrogenase lipoamide kinase isozyme 4（PDK4）（P〈0.01）,and also siginificantly up-regulated the expression of sterol regulatory element binding protein-1c（SREBP1c）,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-103-1down-regulated the expression of PANK3,and improved the mRNA level of SREBP1 c by feedback regulation,finally promoted the de novo synthesis of fatty acid from ACACApa

关 键 词：水牛 bbu-miR-103-1 表达模式 PANK3 

分 类 号：S823.83[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]