Retrotransposon is an important component of the mammalian genome. Previous studies have shown that the expression of protein-coding genes was affected by the insertion of retrotransposon into the proximal genes, and the phenotype variations would be related to the retrotransposon insertion polymorphisms (RIPs). In this study, leptin (LEP), leptin receptor (LEPR), and leptin receptor overlapping transcript (LEPROT), which play important roles in the regulation of fat synthesis and body weight, were screened to search for the RIPs and their effect on phenotype and gene expression, as well as to further study the function of the insertion. The results showed that three RIPs located in intron 1 of LEPROT and intron 2 and 21 of LEPR were identified, and they were all SINEA1, which was one type of retrotransposon. The SINE insertion at the LEPROT was the dominant allele in native pig breeds. The age of 100 kg body weight of SINE+/+ Large White individuals was significantly higher than those of SINE+/− and SINE−/− individuals (p < 0.05). The LEPROT gene expression in the liver and suet of 30-day-old SINE−/− Sujiang piglets were significantly higher than those of SINE+/+ and SINE+/− piglets (p < 0.01). The dual-luciferase reporter gene assay showed that SINE insertion in PK15 and 3T3-L1 cells significantly reduced the promoter activity of the LEPROT gene (p < 0.01). Therefore, SINE insertion can be a repressor to reduce the expression of LEPROT and could be a useful molecular marker for assisted selection of growth traits in pig breeding.
Background: Insulin-like growth factor binding proteins (IGFBPs) have been considered important candidate genes for economic traits due to their involvement in physiological processes related to growth and development. However, most of the current studies on genetic markers of IGFBPs have focused on SNPs, and large fragment insertion mutations such as retrotransposons have rarely been considered.Results: In total twelve retrotransposon insertion polymorphisms (RIPs) were confirmed using bioinformatics prediction combined with the PCR-based amplification. By linkage genetic analysis, IGFBP3-1-RIP and IGFBP3-2-RIP are completely linked, showing only three genotypes, SINE+/+/LINE-/-, SINE-/-/LINE+/+ and SINE+/-/LINE-/+. The age of 100 kg body weight and longissimus muscle thickness of Large white individuals of SINE+/+/LINE-/-genotype were significantly (P<0.05) higher than those of other two genotypes. However, corrected backfat thickness of SINE+/+/LINE-/- individuals were significantly (P<0.05) thinner than those of SINE+/+/LINE-/- genotype. The expression of the IGFBP3 gene in liver and backfat of 30-day Sujiang piglets with SINE+/+/LINE-/- genotype were significantly higher (P<0.05) than those with SINE-/-/LINE+/+ genotype by qPCR. After the core promoter region of the IGFBP3 gene was identified locating within 482bp upstream of ATG by using the dual-luciferase activity assay, further study was conducted to confirm the effect of SINE of IGFBP3-1-RIP and LINE of IGFBP3-2-RIP on the promoter activity of IGFBP3 based on the PGL3-Promoter-Enhancer. The result revealed that only SINE insertion was significantly increased (P<0.05) promoter activity of the IGFBP3 gene, indicating that the SINE may act as an enhancer to regulate the promoter activity of the IGFBP3 gene.Conclusions: Overall, this study identified 12 RIPs in IGFBP gene clusters. Furthermore, SINE insertions in 5’ flanking of IGFBP3 may associated with variations of gene expression and phenotype.
Background: Insulin-like growth factor binding proteins (IGFBPs), specifically binding to IGF1 and IGF2, play an important role in regulating physiological functions of insulin-like growth factors (IGFs). IGFBPs have been considered important candidate genes for economic traits due to their involvement in physiological processes related to growth and development. However, most of the current studies on genetic markers of IGFBPs have focused on SNPs, and large fragment insertion mutations such as retrotransposons have rarely been considered. In this paper, we screened the porcine IGFBP genes (IGFBP1-8) for retrotransposon insertion polymorphisms (RIPs) using bioinformatics prediction combined with the PCR-based amplification. Furthermore, for two linked RIPs their population distribution and impact on promoter activity and phenotype were further evaluated.Results: Screening of IGFBPs identified RIPs in IGFBP1-5 and IGFBP7. In total twelve predicted RIPs were confirmed by PCR. These RIPs were detected in different breeds with an uneven distribution among them. By linkage genetic analysis and PCR verification, IGFBP3-1-RIP and IGFBP3-2-RIP are completely linked, showing only three genotypes, SINE+/+/LINE-/-, SINE-/-/LINE+/+ and SINE+/-/LINE-/+. The age of 100 kg body weight and longissimus muscle thickness of Large white individuals of SINE+/+/LINE-/-genotype were significantly (P<0.05) higher than those of SINE+/-/LINE+/- genotype and SINE-/-/LINE+/+ genotype. However, the longissimus muscle thickness and corrected backfat thickness of SINE+/+/LINE-/- individuals were significantly (P<0.05) thinner than those of SINE+/+/LINE-/- genotype. The expression of the IGFBP3 gene in liver, leg muscles and backfat of 30-day Sujiang piglets with SINE+/+/LINE-/- genotype were significantly higher (P<0.05) than those with SINE-/-/LINE+/+ genotype by the real-time quantitative polymerase chain reaction (qPCR). Further study was conducted to confirm the effect of SINE and LINE insertion on the promoter activity of IGFBP3. First, the core promoter region of the IGFBP3 gene was identified locating within 482bp upstream of ATG by using the dual-luciferase activity assay. Then SINE and LINE were combined with 482bp fragment to construct a recombinant vector respectively based on the PGL3-Promoter-Enhancer. The recombinant vector was transfected into C2C12, 3T3-L1, and Hela cells. The detection of the dual-luciferase reporter gene revealed that only SINE insertion was significantly increased (P<0.05) promoter activity of the IGFBP3 gene, indicating that the SINE may act as an enhancer to regulate the promoter activity of the IGFBP3 gene. Conclusions: Overall, this study identified 12 RIPs in IGFBP gene clusters, which provided useful markers for genetic analysis of pig populations. Furthermore, based on the dual-luciferase activity assay in cells and association analysis, the linked genetic variations generated by SINE and LINE insertions in 5’ flanking of IGFBP3 may associated with variations of gene expression and phenotype.
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