水牛SCAP和SREBP1基因的克

发布时间：2019-05-24 00:41

【摘要】：固醇调节元件结合蛋白裂解激活蛋白(SCAP)是位于内质网上的一种膜结合蛋白,具有一个固醇感受区域,能感应细胞内胆固醇水平的变化；固醇调节元件结合蛋白1(SREBP1)是内质网上一种膜结合转录因子,受SCAP刺激后能激活一系列与脂肪酸合成相关酶的转录表达。SCAP和SREBP1共同维持细胞内固醇水平的稳态,在生物体脂质代谢过程中发挥着重要的作用。本研究以水牛SCAP和SRBEP1为研究对象,初步探讨其与水牛产奶性能的关系,首先进行基因的克隆并对其进行生物信息学分析；其次研究水牛SCAP和SREBP1基因在不同组织、不同泌乳期乳腺组织和高低产水牛奶中的表达；最后采用DNA直接测序方法检测SCAP、SREB1基因的多态性位点,为下一步根据这些基因的多态位点筛选出与水牛产奶性状相关的SNPs标记打好基础,本研究主要结果如下：1. SCAP、SREBP1基因的克隆根据Genbank上公布的牛SCAP基因(NC_007320)和SREBP1基因(NC_007317)设计引物进行克隆,经测序得到4214 bp的SCAP基因mRNA序列4214 bp,其开放阅读框为3837 bp,和3961 bp的SREBP1基因mRNA序列,其编码序列长3438 bp；分析水牛SCAP和SREBP1与牛、人、猪、绵羊、马、小鼠、大鼠、兔和狗CDS同源性分别为99%、98%、89%、96%、89%、85%、85%、87%、89%和98%、86%、88%、96%、87%、79%、80%、82%、86%,并构建进化树分析得到水牛与牛进化距离最近,说明SCAP和SREBP1在不同物种间具有保守性。2. SCAP、SREBP1基因的mRNA表达水平研究以GAPDH为参考基因,提取水牛心、肺、肾、上皮、脾、卵巢、乳腺、脑、大肠、小肠、瘤胃、皱胃、瓣胃、肌肉、脂肪、淋巴、肝、胰组织,泌乳7d、50d、280d水牛乳腺组织,高产和低产水牛奶的RNA作为模板进行实时荧光定量PCR,结果：(1)不同组织中,SCAP基因在乳腺中表达量最高,在淋巴中表达量最低；SREBP1基因在乳腺组织中的表达量最高,在肌肉中表达量最低,说明SCAP和SREBP1基因与水牛乳腺的功能相关；(2)不同泌乳期乳腺中,SCAP基因的表达量为7 d50 d280 d,SREBP1基因的表达量为50 d7 d280 d,说明SCAP基因对水牛泌乳的发动有一定的促进作用,SREBP1基因能促进水牛乳腺泌乳；(3)SCAP和SREBP1基因在高产水牛奶中的表达量均明显高于低产水牛奶,说明SCAP和SREBP1对水牛泌乳量有影响。由此可推测SCAP和SREBP1基因与水牛产奶性状相关联。3. SCAP、SREBP1基因多态性位点的检测PCR扩增18头水牛血液基因组DNA,产物直接测序检测基因多态位点。得到SCAP基因共30个SNPs,25个位于内含子区、5个位于外显子区,其中有1个错义突变、4个同义突变；SREBP1基因共23个SNPs,14个位于内含子区,9个位于外显子区,其中有5个错义突变、4个同义突变。
[Abstract]:Sterol regulatory element binding protein lyase activating protein (SCAP) is a membrane binding protein located in endoplasmic reticulum, which has a sterol receptive region and can induce changes in intracellular cholesterol levels. Sterol regulatory element binding protein 1 (SREBP1) is a membrane binding transcription factor in endoplasmic reticulum. Stimulated by SCAP, it can activate the transcriptional expression of a series of enzymes related to fatty acid synthesis. Cap and SREBP1 can jointly maintain the homeostasis of intracellular sterol levels. It plays an important role in the process of lipid metabolism in organisms. In this study, buffalo SCAP and SRBEP1 were taken as the research objects, and the relationship between them and milk production performance of buffaloes was discussed. Firstly, the genes were cloned and analyzed by bioinformatics. Secondly, the expression of SCAP and SREBP1 genes in different tissues, different lactation stages and high and low water milk was studied. Finally, DNA direct sequencing method was used to detect the polymorphism of SCAP,SREB1 gene, which laid a good foundation for the next step to screen the SNPs markers related to milk production traits of buffalo according to the polymorphism loci of these genes. The main results of this study are as follows: 1. The cloning of SCAP,SREBP1 gene was cloned according to the bovine SCAP gene (NC_007320) and SREBP1 gene (NC_007317) published on Genbank. The open reading frame of 4214 bp SCAP gene mRNA sequence 4214 bp, was 3837 bp,. And 3961 bp SREBP1 gene mRNA sequence, the coding sequence is 3438 bp;. The homology of buffalo SCAP and SREBP1 with CDS of cattle, human, pig, sheep, horse, mouse, rat, rabbit and dog was 99%, 98%, 89%, 96%, 89%, 85%, 85%, 87%, 89%, 89%, 86%, 96%, 87%, respectively. 79%, 80%, 82%, 86%, and the evolutionary tree analysis showed that the evolution distance between buffaloes and cattle was the closest, indicating that SCAP and SREBP1 were conservative among different species. 2. Study on mRNA expression level of SCAP,SREBP1 gene using GAPDH as reference gene, water buffalo heart, lung, kidney, epithelial, spleen, ovary, breast, brain, large intestine, small intestine, rumen, abomasum, flap stomach, muscle, fat, lymph, liver and pancreas were extracted. The results of real-time fluorescence quantitative PCR, were as follows: (1) among different tissues, the expression of SCAP gene was the highest in mammary gland and the lowest in lymphoid tissue, and the results were as follows: (1) the expression of SCAP gene was the highest in mammary gland and the lowest in lymphoid tissue, and the expression of SCAP gene was the highest in breast tissue and the lowest in lymphoid tissue. The expression of SREBP1 gene was the highest in breast tissue and the lowest in muscle, which indicated that SCAP and SREBP1 genes were related to the function of buffalo breast. (2) in different lactation stages, the expression of SCAP gene was 7 d 50 d 280 d, and the expression of SREBP1 gene was 50 d 7 d 280 d, which indicated that SCAP gene could promote the milk production of buffaloes, and SREBP1 gene could promote milk secretion of buffalo milk. The expression of SREBP1 gene was 7 d 50 d 280 d and 50 d 7 d 280 d, which indicated that SREBP1 gene could promote milk secretion of buffalo milk. (3) the expression of SCAP and SREBP1 genes in high yield water milk was significantly higher than that in low yield water milk, which indicated that SCAP and SREBP1 had an effect on the milk yield of buffalo. It can be inferred that SCAP and SREBP1 genes are associated with milk production traits in buffaloes. Detection of SCAP,SREBP1 gene polymorphism PCR amplification of genomic DNA, products from 18 buffaloes by direct sequencing to detect gene polymorphism sites. A total of 30 SNPs,25 of SCAP gene were located in intron region and 5 in exocrine region, including 1 missense mutation and 4 synonymous mutation. A total of 23 SNPs,14 of SREBP1 gene were located in intron region and 9 in exocrine region, including 5 missense mutations and 4 synonymous mutations.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823.83

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