【作者】 熊琪；

【导师】 邓昌彦；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 猪肌肉组织中能量的贮存、释放、转移和利用是决定肌肉生长发育和猪肉品质的重要因素。水解磷脂酰肌醇(phosphatidyldinositol,PI)的肌醇多磷酸5-磷酸酶家族成员对细胞的糖脂代谢信号和膜运输起重要调节作用。其中骨骼肌和肾脏高表达的5’肌醇磷酸酶(Skeletal muscle and kidney enriched inositol phosphatase, SKIP)和包含SH2结构的肌醇多磷酸5-磷酸酶(SH2-containing inositol polyphosphate 5-phosphatase, SHIP2)都能通过水解胰岛素介导的脂质第二信使PI(3,4,5)P3,负调控P13K依赖的胰岛素信号通路,是影响动物个体生长速度和肉用品质的候选基因。因此,本研究对猪SKIP和SHIP2基因进行了分离和遗传效应分析,研究了它们的表达调控机理,并运用RNAi方法对SKIP的功能进行了验证,取得了以下结果：1采用电子克隆方法获得两个候选基因的cDNA序列,均包括完整的编码区(CDS)序列：(1)SKIP,获得cDNA序列1575 bp,其中CDS 1353 bp, GenBank登录号GQ504265。(2)SHIP2,获得cDNA序列4411 bp,其中CDS 3795 bp, GenBank登录号为GU391030。根据基因组比较图谱将SKIP基因定位于猪12号染色体SSC 12q1.3,将SHIP2基因定位于猪9号染色体SSC 9p23-24。分析系统进化树,发现猪SKIP与牛进化关系较近,猪SHIP2与狗进化关系较近。2采用RCR-RFLP技术,在不同猪种中对2个候选基因的SNP位点进行基因分型,并在大白×梅山F2代群体中进行性状关联分析。结果表明(1)SKIP,第12外显子G136A位点与皮率,至第一颈椎胴体长,至第一肋胸胴体长呈极显著相关(P<0.01),与肌内脂肪,含水率呈显著相关(P<0.05),第6内含子A17G位点与骨率,内脂率,至第一肋胸胴体长呈极显著相关(P<0.01),与至第一颈椎胴体长呈显著相关(P<0.05)；第1内含子C1092T位点与内脂率,至第一肋胸胴体长,股二头肌大理石纹,肌内脂肪含量呈显著相关(P<0.05),与背最长肌大理石纹呈极显著相关(P<0.01)；(2)SHIP2,第21内含子G410A与皮率显著相关(P<0.05),与肩部最厚处背膘厚,眼肌高,眼肌宽呈极显著相关(P<0.01)。3运用RNA干扰技术和超表达技术研究了SKIP在分化肌细胞C2C12中的功能,Western blot分析发现抑制SKIP的表达使胰岛素介导的Akt(蛋白激酶B)的磷酸化水平、GSK-3β(糖原合酶激酶)的磷酸化水平,和GS(糖原合酶)的去磷酸化水平都有显著提高,同时发现当在肌细胞内超表达外源猪SKIP时,胰岛素介导的糖原合成量显著减少。表明在肌细胞中,SKIP对Akt/GSK-3p/GS介导的糖原合成信号通路起负调控作用。同时也解释了SKIP基因多态影响肉质性状的分子机理。4利用基因组PCR步移方法获得猪SKIP基因转录起始位点上游2075bp的启动子序列。生物信息学分析发现两个潜在的MyoD结合位点,若干Spl结合位点和一个位于起始密码子上游的CpG岛。以启动子序列为模板,用PCR方法获得5个5’侧翼缺失片断构建pGL3-basic的双荧光素酶报告基因载体,并将这些载体分别转染C2C12成肌分化前体细胞和肌管细胞,荧光素酶活性分析推测在启动子的-1845到-1171和-1171到-737两处地方存在转录抑制元件,在-2038到-1845处存在潜在的转录增强子。5半定量分析发现,SKIP在成年大白猪肌肉组织中高表达,并且在骨骼肌细胞的分化过程中表达上调。利用定点突变和RNA干扰技术,分析了SKIP启动子中潜在E-box元件和转录因子MyoD对SKIP转录的影响。发现突变E-box元件和抑制MyoD的表达都显著降低了肌管细胞中SKIP启动子的活性,表明MyoD可能通过顺式作用元件E-box介导SKIP在肌管细胞中的上调表达,并且TGF-β也以MyoD依赖的方式调控SKIP的转录。6利用RNA干扰技术分析了转录因子Spl对SKIP的转录影响。定量分析发现Spl基因的表达抑制使SKIP启动子活性显著下降,表明转录因子Spl可能通过顺式作用元件GC-box对肌肉细胞分化过程中SKIP基因的转录激活起正向调控作用。同时,SKIP启动子活性在Spl和MyoD双重表达抑制的肌细胞中与在Spl或MyoD单一表达抑制的肌细胞中相比,差异不显著,表明Spl可能与MyoD家族成员协同激活肌细胞分化过程中SKIP的表达。7利用猪基因组信息,分离获得猪SHIP2基因转录起始位点上游1820 bp的启动子序列。生物信息学分析发现Sp1, E2F, MyoD, E47, E2等转录因子的潜在结合位点和覆盖整个启动子区的CpG岛。以启动子序列为模板,用PCR方法获得5个5’侧翼缺失片断构建pGL3-basic双荧光素酶报告基因载体,并将这些载体分别转染C2C12成肌分化前体细胞和肌管细胞,荧光素酶活性分析推测在启动子的-1849到-1742存在转录抑制元件,在-1742到-1694处存在转录增强元件,从-1742到-1266处476bp序列已足够使SHIP2正常转录。8半定量分析发现SHIP2在成年大白猪的肌肉组织中高表达,定量分析发现SHIP2在骨骼肌细胞的分化早期表达上调而在分化中晚期表达下调。利用RNA干扰技术,分析了转录因子MyoD和Spl对SHIP2转录的影响。MyoD或Spl的表达抑制均显著降低了SHIP2的启动子活性,表明转录因子MyoD和Spl均能促进SKIP启动子在肌管细胞中的表达活性。利用定点突变技术分析了E2F作用元件对SHIP2启动子活性的影响,发现突变的E2F结合元件导致SHIP2启动子在成肌前体细胞中的活性下降,而在肌管细胞中的活性提高。表明转录因子E2F可能具有双向调控作用,在成肌前体细胞中促进SHIP2基因的转录,而在肌管细胞中抑制SHIP2基因的转录。更多还原

【Abstract】 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.更多还原