洋葱果胶甲酯酶基因AcPME的克

发布时间：2018-05-28 13:54

本文选题：洋葱 + AcPME的克隆　；参考：《山东农业大学》2017年硕士论文

【摘要】：洋葱(Allium cepa L.)在分类学上属于天门冬目石蒜科葱亚科葱属(APGIII)蔬菜作物,广泛栽培于世界各地。因其基因组巨大(15,290 Mbp/C),分子生物学基础领域的研究相对薄弱,主要集中在育性、颜色相关基因的分子标记上,而关于洋葱功能基因的研究甚少。果胶甲酯酶[pectin methylesterase,PME]是一类催化果胶复合物中甲酯化的D-半乳糖醛酸单元去甲酯化的一类细胞壁蛋白,根据其保守的结构域该酶组成了一个巨大的基因家族,在植物不同的生长发育阶段及其不同的生理过程中,通过作用于细胞壁而发挥作用。已有的研究发现,果胶甲酯酶参与了多种生理过程,如果实成熟、器官脱落、小孢子发育和花粉管伸长、种子萌发、抗病性、形成层细胞分化等。本试验在研究洋葱育性相关基因差异表达的过程中,发现了一个大小约为350 bp的差异表达片段始终只在可育材料中出现,克隆了其编码序列。蛋白质序列比对显示,该基因表达的蛋白具有果胶甲酯酶结构域,属于果胶甲酯酶超家族成员。本研究以洋葱育性恢复系12-10(S,MsMs)为研究材料,利用基因克隆、序列分析、原核表达、生物活性分析及基因枪技术转化洋葱表皮等方法,从分子生物学实验层面初步证明了洋葱AcPME基因是一个在洋葱花粉发育过程中表达的具有PME蛋白活性的定位于细胞壁或细胞膜的PME蛋白超家族成员。本研究结果如下:(1)以洋葱花蕾为试材,通过RACE实验获得了AcPME基因的表达序列;蛋白同源比对与系统进化分析表明,洋葱PME蛋白与水稻、玉米、高粱等单子叶植物的同源蛋白具有较高的相似性;生物信息学分析表明,AcPME蛋白存在明显的信号肽和跨膜区域(SP/TM)、PMEI结构域和PME结构域,在PME家族分类中属于第一类,即含有一个长N末端前区域(pro-region);AcPME蛋白三维结构模式图显示了该蛋白具有一个明显的凹沟,4个结合位点氨基酸残基(T423、Q453、R565和W567),2个活性位点氨基酸残基(D476和D497)。(2)成功构建了pGEX4T-1-PME和pET24a-PMEI原核表达载体,并在大肠杆菌BL21(DE3)菌株中进行了表达条件的优化,确定了pGEX4T-1-PME表达条件为17 oC,0.3 mM IPTG诱导4 h;pET24a-PMEI蛋白表达条件为37 oC,0.1 mM IPTG诱导4 h。SDS-PAGE电泳检测明确了重组蛋白的存在形式,原核表达蛋白pGEX4T-1-PME为可溶性蛋白,而pET24a-PMEI蛋白则以包涵体形式存在;表达蛋白对应的分子量分别约为61.73 kD和24.67 kD,与目的蛋白预期大小相符。(3)利用GST-tag纯化柱对pGEX4T-1-PME表达产物纯化了目的蛋白,同时利用His-tag纯化柱采用柱上复性的方法纯化了目的蛋白pET24a-PMEI,获得了GST-PME和PMEI-6×His纯化蛋白。(4)生物活性分析表明pGEX4T-1-PME(PME)具有明显的PME活性;pET24a-PMEI(PMEI)能够抑制自身蛋白PME的活性,而不能抑制外源蛋白P5400的活性。(5)利用pUC19载体和Ac GFP(Clontech)绿色荧光蛋白基因,成功构建了pUC19-35S-AcPME-AcGFP亚细胞定位载体,并以pUC19-35S-AcGFP载体为阳性对照,利用基因枪轰击洋葱表皮方法进行亚细胞定位,在荧光显微镜下观察发现AcPME融合蛋白定位于细胞膜或细胞壁中,初步证明了该蛋白是一种作用于细胞膜或细胞壁蛋白。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S633.2

【参考文献】