本文選題：草魚 + TGF-β1　； 參考：《上海海洋大學(xué)》2017年碩士論文

【摘要】：草魚(Ctenopharyngodon idella)是目前全球最大的淡水養(yǎng)殖品種,僅在國內(nèi)年產(chǎn)量就4百余萬噸,它不僅是世界上發(fā)達地區(qū)和欠發(fā)達地區(qū)廉價蛋白的主要來源,同時也為優(yōu)質(zhì)動物蛋白的供應(yīng)做出了巨大貢獻[1]。關(guān)于草魚營養(yǎng)學(xué)研究也早已開始,針對目前的研究現(xiàn)狀我們也做了一些營養(yǎng)調(diào)控機理方面的探索,先前有學(xué)者在研究工作中發(fā)現(xiàn)攝食蠶豆的草魚肌肉硬度顯著增加而深受消費者歡迎[2];有研究顯示魚類肌肉硬度的增加與Ⅰ型膠原蛋白的表達有顯著相關(guān)性[3],而Ⅰ型膠原蛋白的表達受TGF-β1/Smad4信號通路調(diào)控[4];為了探究草魚肌肉品質(zhì)改良的營養(yǎng)調(diào)控機制,本文主要構(gòu)建了草魚TGF-β1/Smad4信號通路關(guān)鍵信號因子TGF-β1和Smad4基因的真核過表達載體pcDNA3.1(+)-TGF-β1和pcDNA3.1(+)-Smad4和RNA干擾表達載體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),隨后分別在細胞和魚體水平驗證構(gòu)建的過表達和RNA干擾表達載體的活性,企圖為后續(xù)研究奠定堅實的理論基礎(chǔ),現(xiàn)將本文主要實驗內(nèi)容概括如下:1、本文首先克隆了草魚TGF-β1、Smad4基因開放閱讀框(ORF,Open Reading Frame)序列各1134 bp、1644 bp。其次,在TGF-β1、Smad4序列兩端分別加上相應(yīng)的酶切位點,同時對pcDNA3.1(+)真核表達載體進行雙酶切,將帶酶切位點的片段正向插入到真核表達載體pcDNA3.1(+)中,構(gòu)建TGF-β1、Smad4基因的真核過表達載體pcDNA3.1(+)-TGF-β1、pcDNA3.1(+)-Smad4.另一方面,根據(jù)TGF-β1、Smad4基因序列全長分別設(shè)計3對長度為48 bp的shRNA,然后將構(gòu)建好的shRNA插入到載體pRNA-U6.1/Neo中,即成功構(gòu)建TGF-β1、Smad4基因的RNA干擾表達載體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)。本文構(gòu)建成功的草魚TGF-β1、Smad4基因的RNA干擾表達載體和真核過表達載體,可為下一步草魚肌肉發(fā)育過程中TGF-β1/smad4信號通路作用的研究奠定基礎(chǔ)。2、為在細胞水平上進一步檢測構(gòu)建的過表達和RNA干擾表達載體的有效性,本文首先進行了草魚成纖維細胞的分離和原代培養(yǎng),因分離后的草魚成纖維細胞未能形成穩(wěn)定細胞系,隨后將過表達載體和干擾載體轉(zhuǎn)染至穩(wěn)定的斑馬魚ZF4細胞系;因轉(zhuǎn)染效果不佳,后期針對不同物種做了相關(guān)的生物信息學(xué)分析(包括蛋白結(jié)構(gòu)域和氨基酸序列同源性比對),同源性比對結(jié)果顯示人與草魚蛋白序列同源性高達百分之九十以上;隨后將過表達載體和RNA干擾表達載體轉(zhuǎn)染至293T細胞做進一步驗證實驗,轉(zhuǎn)染36h(轉(zhuǎn)染效率可達80%)后熒光定量檢測TGF-β1、Smad4和Ⅰ型膠原蛋白(COL1-A1和COL1-A2)基因的相對表達量;結(jié)果顯示,過表達實驗組草魚TGF-β1、Smad4及Ⅰ型膠原蛋白(COL1-A1和COL1-A2)基因的表達量顯著高于對照組(P0.05),對人TGF-β1、Smad4和Ⅰ型膠原蛋白(COL1-A1和COL1-A2)基因的檢測結(jié)果顯示無顯著差異;RNA干擾實驗組相比于對照組Ⅰ型膠原蛋白(COL1-A1和COL1-A2)及TGF-β1、Smad4基因的表達量較對照組顯著降低(P0.05)且鑒定出pRNA-6.1/Neo-TGF-β1(Ⅲ)和pRNA-6.1/Neo-Smad4(Ⅱ)的干擾效果最好.本實驗已在細胞水平上很好的驗證了表達載體的有效性。3、本文在魚體水平又進一步的檢測了TGF-β1、Smad4基因過表達和RNA干擾表達載體的活性,結(jié)果顯示過表達實驗組36h和48h目的基因相對表達量顯著高于對照組;RNA干擾表達載體pRNA-6.1/Neo-TGF-β1(Ⅲ)和pRNA-6.1/Neo-Smad4(Ⅱ)36h均有一定的干擾效果但效果不顯著,48h表達量急劇上升,魚體水平的結(jié)果也說明了過表達載體的活性很高,RNA干擾表達載體有一定活性但活性較低;本實驗在魚體水平也較好的驗證了TGF-β1和Smad4基因過表達載體和RNA干擾表達載體的活性。本實驗所進行的TGF-β1和Smad4基因過表達和RNA干擾表達載體的構(gòu)建及其活性驗證,不僅可為后期從營養(yǎng)學(xué)的角度進一步探究草魚肌肉發(fā)育的營養(yǎng)調(diào)控機制奠定理論基礎(chǔ),也將為其他魚類研究提供參考和借鑒。
[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.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S917.4

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