绵羊ANGPTL4基因克
发布时间：2018-09-08 12:06
【摘要】：血管生成素样蛋白4(ANGPTL4)兼有促进脂肪沉积和脂肪酸氧化的作用。一方面,它通过抑制脂蛋白脂肪酶活性而抑制脂肪水解,导致脂代谢相关疾病的发生。另一方面,通过加强儿茶酚胺或糖皮质激素的活性而促进脂肪分解。目前,对于该蛋白及其编码基因的研究已有报道,但在绵羊中未见报道。本研究在DNA.RNA和蛋白三个水平上对ANGPTL4基因的结构和功能,mRNA和蛋白的表达以及ANGPTL4基因5’非编码区的多态性进行了研究,并与相关性状进行了关联分析。利用RT-PCR技术克隆了绵羊ANGPTL4基因的编码区序列(coding sequence, CDS),用生物信息学方法分析了CDS区及其编码的氨基酸序列。以具有显著脂尾型差异的脂尾型绵羊：广灵大尾羊和小尾寒羊,共96只为研究对象,用实时荧光定量PCR(Quantitative real-time PCR)技术,检测了ANGPTL4 mRNA在2、4、6、8、10、12,共6个年龄组的7种脂肪组织(大网膜、小网膜、皮下、腹膜后、’肾周、肠系膜以及尾部)中的相对表达量,并与两品种的尾性状和屠宰性状进行了关联研究。用Western blotting技术检测了ANGPTL4蛋白在两品种母羊的4种脂肪组织(皮下、腹膜后、肾周以及尾部)中的相对表达量。用PCR-SSCP技术检测了96只个体的ANGPTL4基因5’非编码区的单核苷酸多态性(SNP),并与相关性状进行了关联研究。主要结果如下：(1)绵羊ANGPTL4基因CDS区长1233 bp,编码410个氨基酸。预测的ANGPTL4蛋白的等电点为9.02,属于碱性蛋白,相对分子量45.5 kDa。该蛋白是一种不稳定的亲水蛋白。作为一个分泌蛋白,它含有一个长为24 AA的信号肽,3次跨膜。氨基酸序列中有一个N-糖基化位点和一个O-糖基化位点,18个磷酸化位点。进化分析表明,绵羊与牛的亲缘关系最近。(2)ANGPTL4 mRNA在广灵大尾羊和小尾寒羊两品种中存在时空表达差异。品种、月龄、组织对ANGPTL4 mRNA表达影响极显著(P0.001)。在广灵大尾羊中的表达量显著高于小尾寒羊。10月龄时的表达量显著高于其它月龄,浅层脂肪组织中的表达量高于在深层脂肪组织。性别影响不显著但是公羊表达量稍低于母羊。品种与月龄(P0.001)、品种与组织(P0.01)和性别与组织(P0.01)间的互作也影响ANGPTL4mRNA的表达。mRNA表达量与屠宰和尾型性状之间的相关性因品种差异而不同。广灵大尾羊各组织中的表达量均与性状呈正相关,其中,皮下、腹膜后、大网膜和小网膜中的表达量与各性状的相关显著。小尾寒羊大网膜和腹膜后脂肪中的表达量与大多数性状呈负相关,其余为正相关。(3)品种、月龄和组织对ANGPTL4蛋白的相对表达量影响极显著(P0.001)。品种与月龄的互作对该蛋白的表达也有显著影响(P0.01)。ANGPTL4蛋白的表达与其mRNA的表达总体趋势一致。(4)检测到ANGPTL4基因5'非编码区的8个SNPs中3个为颠换,5个为转换。显著的品种间差异发生在-1691 CG和-577 TG中。-2361 AG,-2288 GC和-1855AG的等位基因频率品种间差异不显著,但在广灵大尾羊中检测到3种基因型,而在小尾寒羊中只检测到两种基因型。-1691 CG和-1625 GA均以正向超显性遗传方式影响两个品种的尾长、宰前体重和胴体重,以负向超显性效应方式影响广灵大尾羊的屠宰率。
[Abstract]:Angiopoietin-like protein 4 (ANGPTL4) promotes both lipid deposition and fatty acid oxidation. On the one hand, it inhibits lipid hydrolysis by inhibiting lipoprotein lipase activity, leading to lipid metabolism-related diseases. On the other hand, it promotes lipolysis by enhancing the activity of catecholamines or glucocorticoids. The structure and function of ANGPTL4 gene, the expression of mRNA and protein, and the polymorphism of the 5'non-coding region of ANGPTL4 gene were studied at three levels of DNA, RNA and protein, and the correlation between the polymorphism and the related traits was analyzed. The coding sequence (CDS) of sheep ANGPTL4 gene was analyzed by bioinformatics. 96 fat-tailed sheep with significant fat-tail difference, Guangling large-tailed sheep and Small-tailed Han sheep, were selected as the study objects. Quantitative real-time PCR (Quantitative real-time PCR) was used. The relative expression levels of ANGPTL4 mRNA in 7 adipose tissues (omentum, omentum, subcutaneous, retroperitoneal,'perirenal, mesenteric and caudal) of 6 age groups were detected and correlated with tail traits and slaughter traits of two breeds. The ANGPTL4 protein in two breeds of ewes was detected by Western blotting. Relative expression levels in adipose tissue (subcutaneous, retroperitoneal, perirenal, and caudal) were measured by PCR-SSCP. Single nucleotide polymorphisms (SNPs) in the 5'-noncoding region of the ANGPTL4 gene were detected in 96 individuals and correlated with related traits. The main results were as follows: (1) The CDS region of the sheep ANGPTL4 gene was 1233 BP long, encoding 410 amino acids. As a secretory protein, it contains a signal peptide with a length of 24 AA and three transmembranes. The amino acid sequence contains one N-glycosylation site, one O-glycosylation site and 18 phosphorylation sites. The results showed that the relationship between sheep and cattle was the closest. (2) The expression of ANGPTL4 mRNA in Guangling large-tailed sheep and Small-tailed Han sheep was different in time and space. The expression of ANGPTL4 mRNA in superficial adipose tissue was higher than that in deep adipose tissue at the age of 0.01. The correlation between the expression of ANGPTL4 mRNA and slaughter and tail type traits was also affected by the interaction between breed and age (P 0.001), breed and tissue (P 0.01) and sex and tissue (P 0.01). The expression of omentum and retroperitoneal fat was negatively correlated with most traits, and the other traits were positively correlated with breed, age and group. The expression of ANGPTL4 protein was also significantly affected by the interaction between varieties and ages (P 0.01). The overall trend of expression of ANGPTL4 protein was consistent with that of its mRNA. (4) Three of the eight SNPs in the 5'non-coding region of ANGPTL4 gene were found to be transposed and five were transposed. Allele frequencies of - 2361 AG, - 2288 GC and - 1855 AG were not significantly different among breeds, but three genotypes were detected in Guangling large tail sheep and only two genotypes were detected in small tail Han sheep. - 1691 CG and - 1625 GA all affected tail length, pre-slaughter weight and carcass by positive superdominant inheritance. Body weight affected the slaughter rate of Guangling large tail sheep by negative overdominance effect.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826;Q78
本文编号：2230469
[Abstract]:Angiopoietin-like protein 4 (ANGPTL4) promotes both lipid deposition and fatty acid oxidation. On the one hand, it inhibits lipid hydrolysis by inhibiting lipoprotein lipase activity, leading to lipid metabolism-related diseases. On the other hand, it promotes lipolysis by enhancing the activity of catecholamines or glucocorticoids. The structure and function of ANGPTL4 gene, the expression of mRNA and protein, and the polymorphism of the 5'non-coding region of ANGPTL4 gene were studied at three levels of DNA, RNA and protein, and the correlation between the polymorphism and the related traits was analyzed. The coding sequence (CDS) of sheep ANGPTL4 gene was analyzed by bioinformatics. 96 fat-tailed sheep with significant fat-tail difference, Guangling large-tailed sheep and Small-tailed Han sheep, were selected as the study objects. Quantitative real-time PCR (Quantitative real-time PCR) was used. The relative expression levels of ANGPTL4 mRNA in 7 adipose tissues (omentum, omentum, subcutaneous, retroperitoneal,'perirenal, mesenteric and caudal) of 6 age groups were detected and correlated with tail traits and slaughter traits of two breeds. The ANGPTL4 protein in two breeds of ewes was detected by Western blotting. Relative expression levels in adipose tissue (subcutaneous, retroperitoneal, perirenal, and caudal) were measured by PCR-SSCP. Single nucleotide polymorphisms (SNPs) in the 5'-noncoding region of the ANGPTL4 gene were detected in 96 individuals and correlated with related traits. The main results were as follows: (1) The CDS region of the sheep ANGPTL4 gene was 1233 BP long, encoding 410 amino acids. As a secretory protein, it contains a signal peptide with a length of 24 AA and three transmembranes. The amino acid sequence contains one N-glycosylation site, one O-glycosylation site and 18 phosphorylation sites. The results showed that the relationship between sheep and cattle was the closest. (2) The expression of ANGPTL4 mRNA in Guangling large-tailed sheep and Small-tailed Han sheep was different in time and space. The expression of ANGPTL4 mRNA in superficial adipose tissue was higher than that in deep adipose tissue at the age of 0.01. The correlation between the expression of ANGPTL4 mRNA and slaughter and tail type traits was also affected by the interaction between breed and age (P 0.001), breed and tissue (P 0.01) and sex and tissue (P 0.01). The expression of omentum and retroperitoneal fat was negatively correlated with most traits, and the other traits were positively correlated with breed, age and group. The expression of ANGPTL4 protein was also significantly affected by the interaction between varieties and ages (P 0.01). The overall trend of expression of ANGPTL4 protein was consistent with that of its mRNA. (4) Three of the eight SNPs in the 5'non-coding region of ANGPTL4 gene were found to be transposed and five were transposed. Allele frequencies of - 2361 AG, - 2288 GC and - 1855 AG were not significantly different among breeds, but three genotypes were detected in Guangling large tail sheep and only two genotypes were detected in small tail Han sheep. - 1691 CG and - 1625 GA all affected tail length, pre-slaughter weight and carcass by positive superdominant inheritance. Body weight affected the slaughter rate of Guangling large tail sheep by negative overdominance effect.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826;Q78
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