水牛SCAP和SREBP1基因的克
發(fā)布時間：2019-05-24 00:41
【摘要】：固醇調(diào)節(jié)元件結(jié)合蛋白裂解激活蛋白(SCAP)是位于內(nèi)質(zhì)網(wǎng)上的一種膜結(jié)合蛋白,具有一個固醇感受區(qū)域,能感應細胞內(nèi)膽固醇水平的變化；固醇調(diào)節(jié)元件結(jié)合蛋白1(SREBP1)是內(nèi)質(zhì)網(wǎng)上一種膜結(jié)合轉(zhuǎn)錄因子,受SCAP刺激后能激活一系列與脂肪酸合成相關酶的轉(zhuǎn)錄表達。SCAP和SREBP1共同維持細胞內(nèi)固醇水平的穩(wěn)態(tài),在生物體脂質(zhì)代謝過程中發(fā)揮著重要的作用。本研究以水牛SCAP和SRBEP1為研究對象,初步探討其與水牛產(chǎn)奶性能的關系,首先進行基因的克隆并對其進行生物信息學分析；其次研究水牛SCAP和SREBP1基因在不同組織、不同泌乳期乳腺組織和高低產(chǎn)水牛奶中的表達；最后采用DNA直接測序方法檢測SCAP、SREB1基因的多態(tài)性位點,為下一步根據(jù)這些基因的多態(tài)位點篩選出與水牛產(chǎn)奶性狀相關的SNPs標記打好基礎,本研究主要結(jié)果如下：1. SCAP、SREBP1基因的克隆根據(jù)Genbank上公布的牛SCAP基因(NC_007320)和SREBP1基因(NC_007317)設計引物進行克隆,經(jīng)測序得到4214 bp的SCAP基因mRNA序列4214 bp,其開放閱讀框為3837 bp,和3961 bp的SREBP1基因mRNA序列,其編碼序列長3438 bp；分析水牛SCAP和SREBP1與牛、人、豬、綿羊、馬、小鼠、大鼠、兔和狗CDS同源性分別為99%、98%、89%、96%、89%、85%、85%、87%、89%和98%、86%、88%、96%、87%、79%、80%、82%、86%,并構(gòu)建進化樹分析得到水牛與牛進化距離最近,說明SCAP和SREBP1在不同物種間具有保守性。2. SCAP、SREBP1基因的mRNA表達水平研究以GAPDH為參考基因,提取水牛心、肺、腎、上皮、脾、卵巢、乳腺、腦、大腸、小腸、瘤胃、皺胃、瓣胃、肌肉、脂肪、淋巴、肝、胰組織,泌乳7d、50d、280d水牛乳腺組織,高產(chǎn)和低產(chǎn)水牛奶的RNA作為模板進行實時熒光定量PCR,結(jié)果：(1)不同組織中,SCAP基因在乳腺中表達量最高,在淋巴中表達量最低；SREBP1基因在乳腺組織中的表達量最高,在肌肉中表達量最低,說明SCAP和SREBP1基因與水牛乳腺的功能相關；(2)不同泌乳期乳腺中,SCAP基因的表達量為7 d50 d280 d,SREBP1基因的表達量為50 d7 d280 d,說明SCAP基因?qū)λＣ谌榈陌l(fā)動有一定的促進作用,SREBP1基因能促進水牛乳腺泌乳；(3)SCAP和SREBP1基因在高產(chǎn)水牛奶中的表達量均明顯高于低產(chǎn)水牛奶,說明SCAP和SREBP1對水牛泌乳量有影響。由此可推測SCAP和SREBP1基因與水牛產(chǎn)奶性狀相關聯(lián)。3. SCAP、SREBP1基因多態(tài)性位點的檢測PCR擴增18頭水牛血液基因組DNA,產(chǎn)物直接測序檢測基因多態(tài)位點。得到SCAP基因共30個SNPs,25個位于內(nèi)含子區(qū)、5個位于外顯子區(qū),其中有1個錯義突變、4個同義突變；SREBP1基因共23個SNPs,14個位于內(nèi)含子區(qū),9個位于外顯子區(qū),其中有5個錯義突變、4個同義突變。
[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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S823.83
本文編號：2484398
[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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S823.83
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