草鱼TGF-β1、Smad4基因真核过表达和RNA干扰表达载体的构建及其活性验证

发布时间：2018-07-11 15:34

本文选题：草鱼 + TGF-β1　；参考：《上海海洋大学》2017年硕士论文

【摘要】：草鱼(Ctenopharyngodon idella)是目前全球最大的淡水养殖品种,仅在国内年产量就4百余万吨,它不仅是世界上发达地区和欠发达地区廉价蛋白的主要来源,同时也为优质动物蛋白的供应做出了巨大贡献[1]。关于草鱼营养学研究也早已开始,针对目前的研究现状我们也做了一些营养调控机理方面的探索,先前有学者在研究工作中发现摄食蚕豆的草鱼肌肉硬度显著增加而深受消费者欢迎[2];有研究显示鱼类肌肉硬度的增加与Ⅰ型胶原蛋白的表达有显著相关性[3],而Ⅰ型胶原蛋白的表达受TGF-β1/Smad4信号通路调控[4];为了探究草鱼肌肉品质改良的营养调控机制,本文主要构建了草鱼TGF-β1/Smad4信号通路关键信号因子TGF-β1和Smad4基因的真核过表达载体pcDNA3.1(+)-TGF-β1和pcDNA3.1(+)-Smad4和RNA干扰表达载体pRNA-6.1/Neo-TGF-β1(Ⅰ)、pRNA-6.1/Neo-TGF-β1(Ⅱ)、p RNA-6.1/Neo-TGF-β1(Ⅲ)、pRNA-6.1/Neo-Smad4(Ⅰ)、pRNA-6.1/Neo-Smad4(Ⅱ)和pRNA-6.1/Neo-Smad4(Ⅲ),随后分别在细胞和鱼体水平验证构建的过表达和RNA干扰表达载体的活性,企图为后续研究奠定坚实的理论基础,现将本文主要实验内容概括如下:1、本文首先克隆了草鱼TGF-β1、Smad4基因开放阅读框(ORF,Open Reading Frame)序列各1134 bp、1644 bp。其次,在TGF-β1、Smad4序列两端分别加上相应的酶切位点,同时对pcDNA3.1(+)真核表达载体进行双酶切,将带酶切位点的片段正向插入到真核表达载体pcDNA3.1(+)中,构建TGF-β1、Smad4基因的真核过表达载体pcDNA3.1(+)-TGF-β1、pcDNA3.1(+)-Smad4.另一方面,根据TGF-β1、Smad4基因序列全长分别设计3对长度为48 bp的shRNA,然后将构建好的shRNA插入到载体pRNA-U6.1/Neo中,即成功构建TGF-β1、Smad4基因的RNA干扰表达载体pRNA-6.1/Neo-TGF-β1(Ⅰ)、pRNA-6.1/Neo-TGF-β1(Ⅱ)、pRNA-6.1/Neo-TGF-β1(Ⅲ)、pRNA-6.1/Neo-Smad4(Ⅰ)、pRNA-6.1/Neo-Smad4(Ⅱ)和pRNA-6.1/Neo-Smad4(Ⅲ)。本文构建成功的草鱼TGF-β1、Smad4基因的RNA干扰表达载体和真核过表达载体,可为下一步草鱼肌肉发育过程中TGF-β1/smad4信号通路作用的研究奠定基础。2、为在细胞水平上进一步检测构建的过表达和RNA干扰表达载体的有效性,本文首先进行了草鱼成纤维细胞的分离和原代培养,因分离后的草鱼成纤维细胞未能形成稳定细胞系,随后将过表达载体和干扰载体转染至稳定的斑马鱼ZF4细胞系;因转染效果不佳,后期针对不同物种做了相关的生物信息学分析(包括蛋白结构域和氨基酸序列同源性比对),同源性比对结果显示人与草鱼蛋白序列同源性高达百分之九十以上;随后将过表达载体和RNA干扰表达载体转染至293T细胞做进一步验证实验,转染36h(转染效率可达80%)后荧光定量检测TGF-β1、Smad4和Ⅰ型胶原蛋白(COL1-A1和COL1-A2)基因的相对表达量;结果显示,过表达实验组草鱼TGF-β1、Smad4及Ⅰ型胶原蛋白(COL1-A1和COL1-A2)基因的表达量显著高于对照组(P0.05),对人TGF-β1、Smad4和Ⅰ型胶原蛋白(COL1-A1和COL1-A2)基因的检测结果显示无显著差异;RNA干扰实验组相比于对照组Ⅰ型胶原蛋白(COL1-A1和COL1-A2)及TGF-β1、Smad4基因的表达量较对照组显著降低(P0.05)且鉴定出pRNA-6.1/Neo-TGF-β1(Ⅲ)和pRNA-6.1/Neo-Smad4(Ⅱ)的干扰效果最好.本实验已在细胞水平上很好的验证了表达载体的有效性。3、本文在鱼体水平又进一步的检测了TGF-β1、Smad4基因过表达和RNA干扰表达载体的活性,结果显示过表达实验组36h和48h目的基因相对表达量显著高于对照组;RNA干扰表达载体pRNA-6.1/Neo-TGF-β1(Ⅲ)和pRNA-6.1/Neo-Smad4(Ⅱ)36h均有一定的干扰效果但效果不显著,48h表达量急剧上升,鱼体水平的结果也说明了过表达载体的活性很高,RNA干扰表达载体有一定活性但活性较低;本实验在鱼体水平也较好的验证了TGF-β1和Smad4基因过表达载体和RNA干扰表达载体的活性。本实验所进行的TGF-β1和Smad4基因过表达和RNA干扰表达载体的构建及其活性验证,不仅可为后期从营养学的角度进一步探究草鱼肌肉发育的营养调控机制奠定理论基础,也将为其他鱼类研究提供参考和借鉴。
[Abstract]:Grass carp (Ctenopharyngodon idella) is the largest freshwater breed in the world at present. Its annual output is more than 4 million tons in the country. It is not only the main source of cheap protein in the developed and underdeveloped areas, but also a great contribution to the supply of high quality animal protein. The research on grass carp nutrition has already begun, as well. In view of the current research situation, we have also done some research on the mechanism of nutrition regulation. In the previous study, some scholars found that the grass carp muscle hardness of the faba bean was greatly increased and the consumer welcomed [2]. Some studies showed that the increase of fish muscle hardness was significantly related to the expression of type I collagen protein, [3], and type I. The expression of collagen is regulated by the TGF- beta 1/Smad4 signaling pathway to regulate [4]. In order to explore the nutritional regulation mechanism of grass carp's muscle quality improvement, this paper mainly constructs the key signal factor TGF- beta 1 of the grass carp TGF- beta 1/Smad4 signaling pathway and the eukaryotic overexpression vector of the Smad4 gene pcDNA3.1 (+) -TGF- beta 1 and pcDNA3.1 (+) -Smad4 and RNA interference expression vectors. -6.1/Neo-TGF- beta 1 (I), pRNA-6.1/Neo-TGF- beta 1 (II), P RNA-6.1/Neo-TGF- beta 1 (III), pRNA-6.1/Neo-Smad4 (I), pRNA-6.1/Neo-Smad4 (II), and pRNA-6.1/Neo-Smad4 (III), followed by cells and fish levels to verify the activity of overexpression and RNA interference expression vector, in an attempt to lay a solid theoretical basis for subsequent research. The main contents of this paper are summarized as follows: 1, this paper first cloned the grass carp TGF- beta 1, the Smad4 gene open reading frame (ORF, Open Reading Frame) sequence 1134 BP, 1644 bp. next, at both ends of TGF- beta 1, Smad4 sequence at both ends of the corresponding enzyme cutting site, simultaneously the pcDNA3.1 (+) eukaryotic expression vector of double enzyme cut, will take the fragment of fragments of the fragment Into the eukaryotic expression vector pcDNA3.1 (+), TGF- beta 1, the eukaryotic overexpression vector of the Smad4 gene, pcDNA3.1 (+) -TGF- beta 1, and pcDNA3.1 (+) -Smad4., respectively, are constructed to construct 3 pairs of shRNA 48 BP respectively according to TGF- beta 1 and the Smad4 gene sequence, and then the constructed shRNA is inserted into the carrier. F- beta 1, Smad4 gene RNA interference expression vector pRNA-6.1/Neo-TGF- beta 1 (I), pRNA-6.1/Neo-TGF- beta 1 (II), pRNA-6.1/Neo-TGF- beta 1 (III), pRNA-6.1/Neo-Smad4 (I), pRNA-6.1/Neo-Smad4 (II) and pRNA-6.1/Neo-Smad4 (III). This paper constructs a successful grass carp TGF- beta 1, Smad4 gene for RNA interference expression vector and eukaryotic overexpression vector. One step of the study of the role of TGF- beta 1/smad4 signaling pathway in the development of grass carp muscle is the basis for the study of.2. In order to further detect the effectiveness of overexpression and RNA interference expression vectors constructed at the cellular level, the isolation and primary culture of the grass carp fibroblasts were first carried out, because the isolated grass carp fibroblasts were not stable. The cell line was fixed and then transfected into a stable zebrafish ZF4 cell line by transfecting over expression vector and interference carrier. Due to poor transfection, the related bioinformatics analysis (including homology of protein domain and amino acid sequence) was done for different species in the later period, and homology showed that the homology of human and grass carp protein sequences was higher than that of the results. More than ninety percent, then transfected to 293T cells by over expression vector and RNA interference expression vector for further verification, and transfection of 36h (the transfection efficiency of up to 80%) was used to detect the relative expression of TGF- beta 1, Smad4 and type I collagen (COL1-A1 and COL1-A2) gene, and the results showed that the over expressed experimental group was grass carp TGF- beta 1, Smad4 and The expression of type I collagen (COL1-A1 and COL1-A2) gene was significantly higher than that of the control group (P0.05). There was no significant difference in the detection results of TGF- beta 1, Smad4 and type I collagen (COL1-A1 and COL1-A2) gene, and the RNA interference group was compared with the control group I collagen egg white (COL1-A1 and COL1-A2) and TGF- beta 1, and the expression of the Smad4 gene was more than that of the control group. The effect of pRNA-6.1/Neo-TGF- beta 1 (III) and pRNA-6.1/Neo-Smad4 (II) was the best. This experiment has proved the effectiveness of the expression vector.3 well at the cell level. This paper further detected the activity of TGF- beta 1, Smad4 gene overexpression and RNA interference expression vector at the fish level. The relative expression of 36h and 48h genes in the experimental group was significantly higher than that of the control group; the RNA interference expression vector pRNA-6.1/Neo-TGF- beta 1 (III) and pRNA-6.1/Neo-Smad4 (II) 36h had some interference effect, but the effect of 48h expression increased sharply. The results of the fish body level also showed that the activity of the overexpressed vector was very high, RNA dry. The activity of TGF- beta 1 and Smad4 gene overexpression vector and RNA interference expression vector is also well verified in the fish body level in this experiment. The overexpression of TGF- beta 1 and Smad4 gene and the construction of RNA interference expression vector and its activity verification in this experiment can not only be used for dietetics in the later period. It will lay a theoretical foundation for further exploring the nutritional regulation mechanism of grass carp's muscle development, and will also provide references for other fish research.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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