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

doi:10.11843/j.issn.0366-6964.2016.11.006

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (11): 2191-2201.doi: 10.11843/j.issn.0366-6964.2016.11.006

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

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*

(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)

收稿日期:2016-01-11 出版日期:2016-11-23 发布日期:2016-11-23
通讯作者: 石德顺，博士，研究员，主要从事动物生殖生理研究，E-mail: ardsshi@gxu.edu.cn；刘庆友，博士，研究员，主要从事动物生殖生理研究，E-mail: qyliu2002@126.com
作者简介:蔡小艳(1982-)，女，山西兴县人，博士生，主要从事动物生殖生理及牧草研发，E-mail: caixiaoyan282@163.com
基金资助:
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）

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

CAI Xiao-yan^1,2， LI Sheng¹，CHEN Qiu-ping¹，WANG Ping¹， DENG Kai¹， LIU Qing-you^1* ，SHI De-shun^1*

（1. State Key Laboratory of Tropical Biological Resources Protection and Utilization, Guangxi University，Nanning 530004，China；2.Guangxi Institute of Animal Science，Nanning 530001，China）

Received:2016-01-11 Online:2016-11-23 Published:2016-11-23

摘要/Abstract

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。

Abstract:

This study aimed to investigate the expression pattern of bbu-miR-103-1 from lactation and non-lactation periods in buffalo (Bubalus bubalis), and to predict its target gene and function. Expression pattern of bbu-miR-103-1 in 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 (HIV). It was packaged and propagated to produce high-titer lentivirus 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. The results showed that the relative expression of bbu-miR-103-1 from lactation period was 5.29 times higher than that from non-lactation period in buffalo（P＜0.01）. The LpEZX-pre-miR-103-1 has been successfully constructed and packaged with the infection titer for 3.47×10⁶ PFU•mL^-1. Overexpress or suppress of bbu-miR-103-1 extremely significantly down-regulated or up-regulated（P＜0.01） the expression level of PANK3 in BMECs. Over expression of bbu-miR-103-1 extremely significantly enhanced the expression of Acetyl-CoA carboxylase alpha (ACACA), Glycerol-3-phosphate acyltransferase 1 mitochondrial (GPAM), Diacylglycerol O-acyltransferase 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-103-1 down-regulated the expression of PANK3，and improved the mRNA level of SREBP1c by feedback regulation，finally promoted the de novo synthesis of fatty acid from ACACA pathway. bbu-miR-103-1 plays an important role in enhancing milk fatty acid synthesis, which provides a molecular base for revealing the mechanism of high-level milk fat content in buffalo.

中图分类号:

蔡小艳，李胜，陈秋萍，王萍，邓凯，刘庆友,石德顺. 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[J]. 畜牧兽医学报, 2016, 47(11): 2191-2201.

CAI Xiao-yan， LI Sheng，CHEN Qiu-ping，WANG Ping， DENG Kai， LIU Qing-you，SHI De-shun. Expression Pattern and Target Regulation Gene of bbu-miR-103-1 from Lactation and Non-lactation Periods in B. bubalis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(11): 2191-2201.

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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

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