miRNA-1296 和miRNA-370对奶山羊MTHFR基因表达的调控作用研究

发布时间：2018-05-02 23:38

本文选题：MTHFR基因 + 碱基突变　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：MTHFR是一种黄素蛋白,在叶酸代谢过程中起到限速作用,而MTHFR的表达可能会影响叶酸代谢继而影响乳蛋白合成。miRNA与靶基因的结合可能会因靶基因3'UTR结合位点处的突变而发生改变,进而影响基因的表达。本课题组前期的研究结果表明,奶山羊MTHFR-3'UTR存在c.2494GA突变。为研究MTHFR基因对乳蛋白合成作用的影响,寻找影响MTHFR基因表达的miRNA显得尤为重要。本研究利用生物信息学分析发现miRNA-1296和mi RNA-370能够与野生型(2494G)MTHFR-3'UTR结合。为了验证c.2494GA突变对miRNA结合位点的影响,本研究克隆了野生型和突变型(2494A)MTHFR-3'UTR,构建野生型和突变型psiCHECK?-2-MTHFR-3′UTR双荧光素酶报告载体。通过脂质体转染法将重组载体和miRNA mimics共转染到乳腺上皮细胞中,检测双荧光素酶的相对表达量。将miRNA mimics单独转染到乳腺上皮细胞中,检测MTHFR蛋白的表达。主要研究结果如下:1、奶山羊MTHFR-3'UTR区的克隆、测序分析及重组载体的构建利用奶山羊基因组克隆出野生型(2494G)和突变型(2494A)MTHFR-3'UTR,全长为314bp。构建野生型和突变型psiCHECK?-2-MTHFR-3′UTR双荧光素酶报告载体,并通过测序和双酶切法鉴定。2、奶山羊乳腺上皮细胞的培养和鉴定采集不同基因型的奶山羊乳腺组织(野生型和突变型),利用组织块贴壁法培养奶山羊乳腺上皮细胞,经过3-5次传代得到纯化的奶山羊乳腺上皮细胞,并对培养的乳腺上皮细胞进行鉴定。3、mi RNA对野生型和突变型重组载体荧光素酶表达的影响将重组载体psiCHECK?-2-MTHFR-3′UTR与miR-370、miR-1296通过脂质体共转染奶山羊乳腺上皮细胞,检测荧光素酶的相对表达量。结果显示,用miRNA-370与重组载体共同转染乳腺上皮细胞时,psiCHECK?-2-MTHFR-3′UTR-2494G荧光素酶活性显著低于psiCHECK?-2-MTHFR-3′UTR-2494A。mi R-1296对重组载体psiCHECK?-2-MTHFR-3′UTR的荧光素酶活性没有影响。结果表明,MTHFR-3'UTR c.2494GA的突变改变了miRNA-370的结合位点。4、miRNA-370对MTHFR基因表达的影响利用Western blot的方法检测MTHFR蛋白在不同基因型乳腺上皮细胞中的表达水平。miRNA-370转染乳腺上皮细胞后,抑制野生型乳腺上皮细胞中MTHFR蛋白的表达,但对突变型没有影响。
[Abstract]:MTHFR is a kind of flavin protein, which plays a rate-limiting role in folic acid metabolism, and the expression of MTHFR may affect folic acid metabolism and then affect the binding of milk protein synthesis. MiRNAs to target gene may be changed by mutation of target gene 3'UTR binding site. And then affect the gene expression. The results of our previous study showed that there was a c.2494GA mutation in dairy goat MTHFR-3'UTR. In order to study the effect of MTHFR gene on the synthesis of lactoprotein, it is very important to find the miRNA that affects the expression of MTHFR gene. In this study, bioinformatics analysis showed that miRNA-1296 and mi RNA-370 could be combined with wild type MTHFR-3UTR. In order to verify the effect of c.2494GA mutation on miRNA binding site, wild and mutant psiCHECK?-2-MTHFR-3'UTR double luciferase reporter vectors were cloned and cloned. The recombinant vector and miRNA mimics were co-transfected into mammary epithelial cells by liposome transfection to detect the relative expression of double luciferase. MiRNA mimics was transfected into breast epithelial cells to detect the expression of MTHFR protein. The main results were as follows: 1. Cloning, sequencing and construction of MTHFR-3'UTR region of dairy goat. The wild type was cloned from the genome of dairy goat, the wild type was cloned from the wild type (2494 G) and the mutant type was identified as MTHFR-3 UTR.The total length of the clone was 314bp. Wild type and mutant psiCHECK?-2-MTHFR-3'UTR double luciferase report vector was constructed. The mammary epithelial cells of dairy goats were identified by sequencing and double enzyme digestion. The mammary gland tissues (wild type and mutant type) of different genotypes were collected and the mammary epithelial cells of dairy goats were cultured by tissue mass adherence method. The purified mammary epithelial cells of dairy goats were obtained after 3-5 passages. The expression of luciferase in wild-type and mutants of mammary epithelial cells was identified. The recombinant vector psiCHECK?-2-MTHFR-3'UTR and miR-370 miR-1296 were co-transfected into dairy goat mammary epithelial cells by liposome. The relative expression of luciferase was detected. The results showed that the luciferase activity of psiCHECK-2-MTHFR-3UUTR-2494G was significantly lower than that of psiCHECK?-2-MTHFR-3'UTR-2494A.mi R-1296 on the luciferase activity of the recombinant vector psiCHECK?-2-MTHFR-3'UTR. The results showed that the luciferase activity of the recombinant vector psiCHECK?-2-MTHFR-3'UTR was significantly lower than that of psiCHECK?-2-MTHFR-3'UTR-2494A.mi R-1296. The results showed that the mutation of MTHFR-3 UTR c.2494GA changed the effect of miRNA-370 binding site .4miRNA-370 on the expression of MTHFR gene. The expression level of MTHFR protein in breast epithelial cells with different genotypes was detected by Western blot method. MiRNA-370 was transfected into mammary epithelial cells. The expression of MTHFR protein in wild-type mammary epithelial cells was inhibited, but no effect on mutant type was observed.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

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