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Molecular Cloning and Characterization of Porcine Intelectin2 Gene

Author: ZhangJuan
Tutor: YangZaiQing
School: Huazhong Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Meishan pigs intelectin2 characterization regulation of gene expression dual-luciferase reporter assay system SREBP-1c
CLC: S828
Type: Master's thesis
Year: 2010
Downloads: 12
Quote: 0
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Meishan breed is famous for its fecundity, it is an important breed resource all over the world. There are still many puzzles on the molecular mechanisms of fat deposition and ectopic fat deposition, lipid metabolism to solve. So functional research of important genes in this breed would not only be necessary for the further genetic improvement in accumulating and exploring the gene resources and breeding, but also for clarifying the molecular mechanisms of fat deposition and ectopic fat deposition, lipid metabolism and so on.Intelectin was first reported as a newly discoved gene which specially expresses in visceral adipose tissue, it cause widespread attention as an adipokine secreted by omental adipose tissue. In humanity, it is the link between obesity, insulin resistance, cardiovascular disease and metabolic syndrome. There are two isoforms:intelectin 1、2, both of the two gene expression were decreased with obesity and were highly correlated with each other in visceral adipose tissue. Besides, as intelectin has a fibrinogen domain, most researches focus on their numerous functions as lectin in intestinal, however, not many more researches on its molecular cloning and characterization in pig.In this study, we first reported here the intelectin2 CDs and 4 introns cloning and tissue distribution by qRT-PCR.We also had successfully cloned and analysised the 5’ flanking region of intelectin2, and further constructed to pGL3 luciferase reporter vector. By using dual luciferase assay system, the activity of the region was analysised in porcine IBRS-2 cells. To understand the expression and regulation mechanism of porcine intelectin2,the promoter pGL3 luciferase reporter vector had been transfected into mouse NIH3T3 with eukaryotic transcription factor pcDNA3.1-C/EBPa (CREB1, KLF15, PPARγ2, SREBP-lc). Dual luciferase assay system was used to detect the activity changes of promoter after transfected transcription factors. Our findings provide the basis for clarifying the regulation method and functions of porcine intelectin2. The main results are as follows:1. We had successfully cloned the intelectin2 CDs(1213bp) and 4 introns (ranging from 234bp to 1047bp).According to the CDs and several introns, we analysis the genomic structure of intelectin2 which consists of 7 introns and 8 exons, all of the exon-intron boundaries are agreed with the GT/AG rule. Porcine intelectin2 share 81%, 84% and 84% identity with intelectin2 gene of human, cattle and mouse, respectively. Tissue distribution by qRT-PCR indicated it is abundant in gut, with low expression in abdominal fat, and undetectable in several tissues.2. Porcine intelectin2 5’flanking region (1389bp) was cloned which has strong promoter activity which proved by dual-luciferase reporter assay system.3. Intelectin2 promoter was constructed into PGL3 dual-luciferase reporter vecter. In order to investigate the expression and regulation mechanism of porcine intelectin2, pcDNA3.1-C/EBPa (CREB1, KLF15, PPARy2, SREBP-lc) were transiently transfected into mouse NIH3T3 cells with promoter PGL3 dual-luciferase reporter vecter, respectively. After transfection, we use the dual-luciferase reporter assay system to test the activity of intelectin2 promoter. The results showed that C/EBPa, CREB1, PPARγ2 and KLF15 had no significant effects on the activity of intelectin2 promoter, on the contrary, SREBP-1c could significant inhibit the activity of porcine intelectrin2 promoter.

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