Cloning, Genetics Effect And Functional Analysis Of Porcine SKIP And SHIP2 Gene

The energy storage, release, transfer and utilization are the key factors in determining skeletal muscle development and pork quality. Inositol polyphosphate 5-phosphatases regulate insulin signaling and intracellular trafficking. Skeletal muscle and kidney enriched inositol phosphatase (SKIP) and SH2-containing inositol polyphosphate 5-phosphatase (SHIP2) identified as 5'-inositol phosphatase family members play a negative role in PI3K-dependent insulin signaling pathway by hydrolysis of PI(3,4,5)P3. As candidate genes of animal growth and meat quality, the porcine SKIP and SKIP were cloned and identified and the promoter regulation and genetics effect were analyzed. By using RNAi technique, we also verified the influence of SKIP on insulin-induced glycogen synthesis signaling. The main results are as follows:1 We obtained the full coding region of the 2 genes with electronic cloning technology. (1)SKIP, obtained cDNA sequence 1575 bp, the CDS is 1353 bp, GenBank accession number. GQ504265. (2) SHIP2, obtained DNA sequence 4411 bp, the CDS is 3795 bp, GenBank accession number GU391030. With the use of comparative mapping, we mapped the SKIP gene to SSC 12q1.3 and the SH1P2 gene to SSC 9p23-24. From the phylogenetic relationship, the porcine protein sequence of SKIP has high sequence similarity with cattle than with other species, and the porcine protein sequence of SHIP2 has high sequence similarity with dog than with other species.2 Using PCR-RFLP, we detected SNPs of 2 genes in different pig populations and observed associations with carcass and meat quality traits in Large White and Meishan F2 hybrids. The results showed:(1) SKIP, G136A-Eco47Ⅰ-RFLP is higher significant association with Skin percentage, Carcass length to 1st spondyle and Carcass length to 1 st rib (P<0.01), high significant association with Intramuscular fat and Water content (LD) (P<0.05); A17G-ApaⅠ-RFLP is higher significant association with Bone percentage, Internal fat rat Carcass length to 1st rib (P<0.01), high significant association with Carcass length to 1st spondyle (P<0.05). C1092T-BamHⅠ-RFLP is higher significant association with Meat marbling (BF) (P<0.01), high significant association with Internal fat rat, Meat marbling (LD) and Intramuscular fat (LD) (P< 0.05) (2) SHIP2, G410A- BamHⅠ-RFLP is higher significant association with Shoulder fat thickness, m.longissimus Dorsi height, and m.longissimus Dorsi width (P< 0.01),high significant association with Skin percentage (P<0.05).3 We employed RNAi technique to study the SKIP function in differentiating C2C12 myoblasts. Western blot analysis showed that insulin-induced phosphorylation of Akt (protein kinase B) and GSK-3β(Glycogen synthase kinase), and dephosphorylation of GS (glycogen synthase) were increased by inhibiting the expression of SKIP, whereas the insulin-induced glycogen synthesis was decreased by overexpression of WT-SKIP. Our results suggest that SKIP plays a negative regulatory role in Akt/GSK-3β/GS (glycogen synthase) pathway leading to glycogen synthesis in myocytes. These results also explained the molecule mechanism of relationship between SKIP polymorphism with meat quality traits.4 Genome walking technique was used to clone the proximal promoter region of porcine SKIP. A fragment of 2075 bp upstream start code was obtained from genomic DNA of porcine. Use computer analyze, tow MyoD binding site, several Sp1 binding site, and a CpG island were found. Based on this fragment, a series of 5'-deletion mutants of SKIP promoter cloned by PCR were cloned upstream of the pGL3-basic constructs and were analyzed using transfection and luciferase activity. Two regions that could inhibit transcription (between -1845 and -1171 and between -1171 and -737) and an area with potential transcriptional enhancers (between -2038 and -1845) were identified in SKIP promoter:5 The tissue expression profile analysis showed that the SKIP gene was expressed at a high level in skeletal muscle. And porcine SKIP was transcriptionally upregulated during skeletal muscle differentiation. Use site-directed mutagenesis and RNAi technique, the effect of E-box element and MyoD on SKIP transcription activity were analyzed. The porcine SKIP promoter activity was significantly decreased by mutated E-box element and inhibiting the expression of MyoD. It demonstrated that MyoD was involved in upregulating SKIP mRNA expression in myotubes, partly via the cis-acting elements in SKIP promoter. SKIP expression is also modulated by TGF-β, and is MyoD dependent.6 Using RNAi technique, the effect of Sp1 on SKIP transcription activity was analyzed. Real-Time PCR analysis indicated a decrease in SKIP transcriptional activity by inhibiting the expression of transcription factor Sp1 in differentiating C2C12 myoblasts. These results suggest that Sp1 plays a positive regulatory role in SKIP expression possibly by GC elements in myotubes. However, there were no significant differences in SKIP promoter activity between myotube cells treated with both Spl-siRNA and MyoD-siRNA and myotube cells treated with only Spl-siRNA or MyoD-siRNA. It was speculated that Sp1 might interact with MyoD family members in regulating SKIP expression during skeletal muscle differentiation.7 Comparative genomic technology was used to clone the proximal promoter region of porcine SHIP2. A fragment of 1820 bp upstream start code was obtained from genomic DNA of porcine. Use computer analyze, a MyoD binding site, a E2F binding site, a E47 binding site, a E2 binding site, several Sp1 binding site, and a large area of CpG island were found. Based on this fragment, a series of 5'-deletion mutants of SHIP2 promoter cloned by PCR were cloned upstream of the pGL3-basic constructs and were analyzed using transfection and luciferase activity. An area that could inhibit transcription (between-1849 and -1742) and an area with potential transcriptional enhancers (between -1742 and-1694) were identified in SHIP2 promoter. The region upstream from position -1742 to-1266 was sufficient for almost intact SHIP2 promoter activity.8 The tissue expression profile analysis showed that the SHIP2 gene was expressed at a high level in skeletal muscle. And porcine SHIP2 was transcriptionally upregulated in early stage and downregulated in intermediate and advanced stages of skeletal muscle differentiation. Use RNAi technique, the effect of MyoD and Sp1 on SHIP2 transcription activity were analyzed. The porcine SHIP2 promoter activity was significantly decreased by inhibiting the expression of both MyoD and Sp1 in differentiating C2C12 myoblasts. It demonstrated that MyoD and Sp1 were involved in upregulating SHIP2 mRNA expression in myotubes. Meanwhile, use site-directed mutagenesis technique, the effect of potential E2F binding element on SKIP transcription activity was analyzed. The luciferase activity of mutated SHIP2 promoter was decreased in myoblast cells and increased in myotube cells. It suggested that transcription factor E2F has a biphase regulation on SHIP2 mRNA expression in different cell types. In other words, the E2F element promotes SHIP2 transcription in myoblasts but inhibits SHIP2 transcription in myotubes.