合浦珠母贝基质蛋白基因的转录调控研究

发布时间：2018-04-19 08:56

本文选题：合浦珠母贝 + 基质蛋白　；参考：《清华大学》2016年博士论文

【摘要】：合浦珠母贝(Pinctada fucata)因盛产珍珠而名,贝壳和珍珠是典型的生物矿化产物。研究表明基质蛋白在合浦珠母贝贝壳和珍珠的形成过程中起着关键的作用,基质蛋白的功能已经得到广泛的研究,但是关于基质蛋白基因的胞内上游转录调控机制知之甚少。本研究从基质蛋白基因启动子的角度着手,结合转录因子的发现和鉴定,来探究合浦珠母贝中基质蛋白基因的上游转录调控机理。在本研究中,合浦珠母贝的Pf-POU2F1和Pf-POU3F4转录因子被克隆和鉴定。同时,我们也克隆得到了基质蛋白基因Aspein和Prismalin-14的启动子序列。细胞共转染的结果表明Pf-POU3F4能显著上调Aspein和Prismalin-14基因的启动子活性并且激活作用具有剂量效应,同时,Pf-POU3F4结构域的完整性对于激活作用是必要的。进一步研究表明,Aspein基因启动子的一个区域(-181~-77 bp)和Prismalin-14基因启动子的两个位点(-359~-337 bp和-100~-73 bp)是Pf-POU3F4的直接结合作用区。活体干扰实验表明敲低Pf-POU3F4的表达量后,Aspein和Prismalin-14基因的表达量随之下调。这些结果揭示了在合浦珠母贝中转录因子Pf-POU3F4能调节基质蛋白基因Aspein和Prismalin-14的表达。此外,基质蛋白基因Shematrin-2、ACCBP、MSI60和N19的启动子序列也得到了克隆和鉴定。其中,Shematrin-2基因启动子的基础活性最高。在探究基质蛋白Shematrin-2功能的同时,对Shematrin-2基因的转录调控也进行了初步的研究。我们克隆得到了三个C/EBP转录因子以及NF-Y转录因子的三个亚基。细胞共转染的结果表明Pf-C/EBP-A和Pf-C/EBP-B能显著上调Shematrin-2基因的启动子活性并且激活作用具有剂量效应。同时,我们也初步探究了Pf-C/EBP-A和Pf-C/EBP-B对其它七种基质蛋白基因启动子活性的影响,结果表明Pf-C/EBP-A和Pf-C/EBP-B能显著上调多种基质蛋白基因的转录活性。这些结果暗示了在合浦珠母贝中转录因子Pf-C/EBP-A和Pf-C/EBP-B可能参与了多种基质蛋白基因的转录调控。我们的研究揭示了调控基质蛋白基因表达相关的转录因子,为进一步研究基质蛋白基因的转录调控机制提供基础,将有助于从分子水平上理解贝壳形成的上游调控机理。
[Abstract]:Pinctada fucata) is famous for producing pearls, shells and pearls are typical biomineralization products.It has been shown that matrix proteins play a key role in the formation of shells and pearls. The function of matrix proteins has been extensively studied, but little is known about the transcriptional regulation mechanism of matrix protein gene upstream.The aim of this study was to explore the upstream transcriptional regulation mechanism of matrix protein gene from the perspective of matrix protein gene promoter combined with the discovery and identification of transcription factors.In this study, Pf-POU2F1 and Pf-POU3F4 transcription factors were cloned and identified.At the same time, we cloned the promoter sequence of matrix protein gene Aspein and Prismalin-14.The results of co-transfection showed that Pf-POU3F4 could significantly up-regulate the promoter activity of Aspein and Prismalin-14 genes and the activation was dose-dependent, and the integrity of the Pf-POU3F4 domain was necessary for the activation.Further studies showed that a region of the Aspein gene promoter (-181- 77bp) and two loci of the Prismalin-14 gene promoter (-359- 337bp and -100- 73bp) were the direct binding regions of Pf-POU3F4.In vivo interference assay showed that the expression of Aspein and Prismalin-14 genes was down-regulated after knock down the expression of Pf-POU3F4.These results suggest that transcription factor Pf-POU3F4 can regulate the expression of matrix protein genes Aspein and Prismalin-14.In addition, the promoter sequences of matrix protein gene Shematrin-2 ACCBPnMSI60 and N19 were also cloned and identified.The basic activity of Shematrin-2 gene promoter was the highest.While exploring the Shematrin-2 function of matrix protein, the transcriptional regulation of Shematrin-2 gene was also studied.We cloned three C/EBP transcription factors and three subunits of NF-Y transcription factors.The results of co-transfection showed that Pf-C/EBP-A and Pf-C/EBP-B could significantly up-regulate the promoter activity of Shematrin-2 gene and the activation was dose-dependent.At the same time, we also investigated the effects of Pf-C/EBP-A and Pf-C/EBP-B on the promoter activity of other seven matrix protein genes. The results showed that Pf-C/EBP-A and Pf-C/EBP-B could significantly up-regulate the transcriptional activity of several matrix protein genes.These results suggest that the transcription factors Pf-C/EBP-A and Pf-C/EBP-B may be involved in the transcriptional regulation of many matrix protein genes.Our research reveals the transcription factors related to the regulation of matrix protein gene expression, which provides the basis for further study on the transcription regulation mechanism of matrix protein gene, and will be helpful to understand the upstream regulation mechanism of shell formation at molecular level.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4

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