本文選題：洋蔥 + AcPME的克隆��； 參考：《山東農業(yè)大學》2017年碩士論文

【摘要】：洋蔥(Allium cepa L.)在分類學上屬于天門冬目石蒜科蔥亞科蔥屬(APGIII)蔬菜作物,廣泛栽培于世界各地。因其基因組巨大(15,290 Mbp/C),分子生物學基礎領域的研究相對薄弱,主要集中在育性、顏色相關基因的分子標記上,而關于洋蔥功能基因的研究甚少。果膠甲酯酶[pectin methylesterase,PME]是一類催化果膠復合物中甲酯化的D-半乳糖醛酸單元去甲酯化的一類細胞壁蛋白,根據其保守的結構域該酶組成了一個巨大的基因家族,在植物不同的生長發(fā)育階段及其不同的生理過程中,通過作用于細胞壁而發(fā)揮作用。已有的研究發(fā)現,果膠甲酯酶參與了多種生理過程,如果實成熟、器官脫落、小孢子發(fā)育和花粉管伸長、種子萌發(fā)、抗病性、形成層細胞分化等。本試驗在研究洋蔥育性相關基因差異表達的過程中,發(fā)現了一個大小約為350 bp的差異表達片段始終只在可育材料中出現,克隆了其編碼序列。蛋白質序列比對顯示,該基因表達的蛋白具有果膠甲酯酶結構域,屬于果膠甲酯酶超家族成員。本研究以洋蔥育性恢復系12-10(S,MsMs)為研究材料,利用基因克隆、序列分析、原核表達、生物活性分析及基因槍技術轉化洋蔥表皮等方法,從分子生物學實驗層面初步證明了洋蔥AcPME基因是一個在洋蔥花粉發(fā)育過程中表達的具有PME蛋白活性的定位于細胞壁或細胞膜的PME蛋白超家族成員。本研究結果如下:(1)以洋蔥花蕾為試材,通過RACE實驗獲得了AcPME基因的表達序列;蛋白同源比對與系統(tǒng)進化分析表明,洋蔥PME蛋白與水稻、玉米、高粱等單子葉植物的同源蛋白具有較高的相似性;生物信息學分析表明,AcPME蛋白存在明顯的信號肽和跨膜區(qū)域(SP/TM)、PMEI結構域和PME結構域,在PME家族分類中屬于第一類,即含有一個長N末端前區(qū)域(pro-region);AcPME蛋白三維結構模式圖顯示了該蛋白具有一個明顯的凹溝,4個結合位點氨基酸殘基(T423、Q453、R565和W567),2個活性位點氨基酸殘基(D476和D497)。(2)成功構建了pGEX4T-1-PME和pET24a-PMEI原核表達載體,并在大腸桿菌BL21(DE3)菌株中進行了表達條件的優(yōu)化,確定了pGEX4T-1-PME表達條件為17 oC,0.3 mM IPTG誘導4 h;pET24a-PMEI蛋白表達條件為37 oC,0.1 mM IPTG誘導4 h。SDS-PAGE電泳檢測明確了重組蛋白的存在形式,原核表達蛋白pGEX4T-1-PME為可溶性蛋白,而pET24a-PMEI蛋白則以包涵體形式存在;表達蛋白對應的分子量分別約為61.73 kD和24.67 kD,與目的蛋白預期大小相符。(3)利用GST-tag純化柱對pGEX4T-1-PME表達產物純化了目的蛋白,同時利用His-tag純化柱采用柱上復性的方法純化了目的蛋白pET24a-PMEI,獲得了GST-PME和PMEI-6×His純化蛋白。(4)生物活性分析表明pGEX4T-1-PME(PME)具有明顯的PME活性;pET24a-PMEI(PMEI)能夠抑制自身蛋白PME的活性,而不能抑制外源蛋白P5400的活性。(5)利用pUC19載體和Ac GFP(Clontech)綠色熒光蛋白基因,成功構建了pUC19-35S-AcPME-AcGFP亞細胞定位載體,并以pUC19-35S-AcGFP載體為陽性對照,利用基因槍轟擊洋蔥表皮方法進行亞細胞定位,在熒光顯微鏡下觀察發(fā)現AcPME融合蛋白定位于細胞膜或細胞壁中,初步證明了該蛋白是一種作用于細胞膜或細胞壁蛋白。
[Abstract]:Onions (Allium CEPA L.) belong to the taxonomy of Allium Allii, asparagus, Allium onions (APGIII), which are widely cultivated in all parts of the world. Because their genome is huge (15290 Mbp/C), the research on the basic field of molecular biology is relatively weak, mainly on the molecular markers of fertility, color related genes, and onions functional genes. There are few studies. Pectin methylesterase [pectin methylesterase and PME] are a class of cell wall proteins that catalyze the methylation of methyl galactoaluronic acid units in pectin complexes. According to their conservative domain, the enzyme consists of a huge gene family in different growth stages and different physiological processes in plants. It has been found that the pectin methylene esterase is involved in a variety of physiological processes, such as real maturation, organ loss, microspore development and pollen tube elongation, seed germination, disease resistance, and formation of cell differentiation. The differentially expressed fragments, about 350 BP, were always found in fertile materials and cloned their coding sequences. The protein sequence alignment showed that the protein expressed in the gene was a domain of pectin methesterase, which belonged to the member of the pectin methesterase superfamily. This study used onion fertility restorer line 12-10 (S, MsMs) as the research material and gene cloning. Sequence analysis, prokaryotic expression, bioactivity analysis and gene gun technology transformation of onion epidermis have proved that the onion AcPME gene is a member of the PME protein superfamily with PME protein activity in the cell wall or cell membrane expressed in the development of onion pollen. The results of this study are the result of this study. As follows: (1) the AcPME gene expression sequence was obtained by RACE experiment with onion bud. The homologous protein of the onion PME protein and the monocotyledon of rice, corn and sorghum had higher similarity. The bioinformatics analysis showed that the AcPME protein had obvious signal peptide and span. The membrane region (SP/TM), the PMEI domain and the PME domain belong to the first class in the PME family classification, which contains a long N terminal region (pro-region); the three-dimensional structure pattern diagram of the AcPME protein shows that the protein has a distinct concave groove, 4 binding sites amino acid residues (T423, Q453, R565 and W567), and the 2 active site amino acid residues (D476). (2) (2) (2) the prokaryotic expression vector of pGEX4T-1-PME and pET24a-PMEI was successfully constructed, and the expression conditions were optimized in Escherichia coli BL21 (DE3) strain. The expression conditions of pGEX4T-1-PME were determined to be 17 oC, 0.3 mM IPTG induced 4 h, and the pET24a-PMEI protein expression condition was 37 oC, and 4 electrophoresis was used to detect the recombinant eggs. In the form of white, prokaryotic expression protein pGEX4T-1-PME is soluble protein, while pET24a-PMEI protein exists in inclusion body form, and the corresponding molecular weight of expressed protein is about 61.73 kD and 24.67 kD, respectively. (3) GST-tag purification column is used to purify the target protein of pGEX4T-1-PME expression product and use H at the same time. Is-tag purification column purified the target protein pET24a-PMEI by the method of refolding on the column and obtained the purified protein of GST-PME and PMEI-6 x His. (4) bioactivity analysis showed that pGEX4T-1-PME (PME) had obvious PME activity; pET24a-PMEI (PMEI) could inhibit the viability of the protein PME, but could not inhibit the activity of exogenous protein P5400. (5) utilization of the protein P5400. The vector and Ac GFP (Clontech) green fluorescent protein gene have successfully constructed the pUC19-35S-AcPME-AcGFP subcellular location vector, and using the pUC19-35S-AcGFP carrier as the positive control, using the gene gun to bombardment of the onion epidermal method for subcellular localization, and observe the localization of the AcPME fusion protein in the cell membrane or cell wall under the fluorescence microscope. It is proved that the protein is a protein that acts on cell membrane or cell wall.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S633.2

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