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

Abstract

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.

CLC Number:

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

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