猪FAM213B基因mRNA和启动子的克隆及序列分析

发布时间：2018-03-12 11:56

本文选题：猪　切入点：FAMB基因　出处：《华南农业大学学报》2017年03期 　论文类型：期刊论文

【摘要】：【目的】获得猪FAM213B基因完整mRNA和启动子序列,研究猪FAM213B基因表达,为探讨母猪妊娠的建立和胚胎发育调控机制奠定基础。【方法】通过5'RACE和3'RACE技术,获得基因完整mRNA序列,分析不同物种该基因氨基酸序列相似性;通过PCR克隆启动子区,并通过双荧光素酶报告基因载体系统转染猪子宫内膜细胞,研究其转录活性。【结果】猪FAM213B基因mRNA全长808 bp,其中5'UTR、CDS区和3'UTR长度分别为67、609(含终止密码子)和132 bp(不含poly A序列),在17~106位氨基酸之间存在硫氧还蛋白折叠结构域;与猪FAM213B基因其他2个潜在转录本相比,三者都包含硫氧还蛋白折叠结构域,但蛋白三级结构存在较大差异;猪FAM213B氨基酸序列与山羊、牛和绵羊高度相似,相似性分别为94.03%、93.03%和91.54%。克隆获得2 261 bp(-2 231/+30)的基因启动子序列,将其连接至双荧光素酶报告基因载体,转染猪子宫内膜细胞,发现获得的启动子片段能够启动下游报告基因的转录,在启动子区存在潜在的典型NFκB等转录因子结合位点。【结论】本研究获得猪FAM213B基因转录本长度为808 bp,其蛋白存在硫氧还蛋白折叠功能结构域,其启动子序列(-2 231/+30)在猪子宫内膜细胞中具有较强的转录活性。
[Abstract]:[objective] to obtain the complete mRNA and promoter sequence of porcine FAM213B gene and study the expression of porcine FAM213B gene in order to lay a foundation for the establishment of pregnancy and the regulation mechanism of embryo development in sows. [methods] the complete mRNA sequence was obtained by 5race and 3RACE techniques. The amino acid sequences of different species were analyzed, the promoter region was cloned by PCR, and the porcine endometrial cells were transfected by double luciferase reporter gene vector system. [results] the total length of porcine FAM213B gene mRNA was 808 BP, in which the length of 5 UTRT CDS region and 3 FAM213B UTR region were 67 / 609 (including termination codon) and 132bp (excluding poly A) respectively, and there was a thioredoxin protein folding domain between the amino acids of 1710 ~ 6 amino acids. Compared with the other two potential transcripts of porcine FAM213B gene, all of them contain thioredoxin folding domain, but there is great difference in the tertiary structure of protein, and the amino acid sequence of porcine FAM213B is highly similar to that of goat, cattle and sheep. The similarity was 94.03% and 91.54%, respectively. The promoter sequence of 2 261 bp(-2 231 / 30) was cloned and ligated into double luciferase reporter gene vector and transfected into porcine endometrial cells. The obtained promoter fragment was found to be able to activate transcription of the downstream reporter gene. There are potential typical NF 魏 B transcription factor binding sites in the promoter region. [conclusion] the length of porcine FAM213B gene transcripts is 808 BP, and the protein has thioredoxin folding functional domain. The promoter sequence of the promoter-2 231 / 30) has a strong transcriptional activity in porcine endometrial cells.
【作者单位】：广东第二师范学院生物与食品工程学院/广东高校应用生态工程技术开发中心;华南农业大学动物科学学院/广东省农业动物基因组学与分子育种重点实验室;
【基金】：国家自然科学基金(31201771) 现代农业产业技术体系专项(CARS-36)
【分类号】：Q78;S828

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