苦荞Ⅱ型金属硫蛋白基因的功能及其启动子活性分析

发布时间：2018-02-12 17:01

本文关键词： 苦荞金属硫蛋白启动子重金属原核表达蛋白纯化　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：金属硫蛋白(metallothioneins,MTs)是一类富含半胱氨酸的低分子量蛋白,在植物重金属耐受性中发挥重要的作用。苦荞(Fagopyrum tataricum)含有丰富的黄酮类物质,是重要的药食两用作物,其具有耐瘠薄、抗重金属等良好的抗逆性。但目前为止,尚未见有关于苦荞重金属抗逆机理和苦荞金属硫蛋白表达调控以及功能的相关研究报道。本研究通过克隆苦荞Ⅱ型金属硫蛋白基因序列(FtMT2),构建FtMT2过表达的大肠杆菌、酵母及拟南芥,并测定重组蛋白的羟自由基去除能力,以期揭示FtMT2在苦荞重金属耐受中的功能;通过FtMT2启动子序列的克隆,预测启动子区的顺式作用元件,构建不同截短启动子的双荧光素酶报告载体,采用瞬时转化苦荞叶肉细胞原生质体研究FtMT2启动子对Cu2+的响应,以期揭示FtMT2基因启动子活性的响应模式。FtMT2的克隆及其启动子的功能分析对于建立Cu2+污染的检测技术具有参考价值,同时为解析苦荞重金属抗性的内在机制奠定了基础。本研究得到的主要结果和结论有:(1)FtMT2基因含有3个外显子和2个内含子分隔,内含子边界符合“5′-GT”和“AG-3′”规则。FtMT2编码区为237 bp,编码78个氨基酸;预测的编码蛋白的分子质量为7.885 kD,等电点4.55,蛋白序列中含有14个Cys,在N-端含有“C-C,C-X-C,C-X-X-C”模体以及高度保守的“MSCCGGNCGCGS”序列,C-端含有3个“C-X-C”模体。N-端和C-端的结构被42个氨基酸的连接区分隔,这些均为植物Ⅱ型金属硫蛋白的典型特征。蛋白序列的系统发育分析显示其与双子叶豆科植物田菁关系最近。(2)生长曲线测定和平板菌落分析结果显示,FtMT2过表达的大肠杆菌TOP10和酿酒酵母分别对Cu2+和Cd2+耐受性增加。暗示FtMT2在苦荞对Cu2+和Cd2+的耐受性中发挥作用。(3)FtMT2重组蛋白的羟自由基去除能力测定结果显示,羟自由基去除率为50%时的蛋白浓度为46.5μg/m L。暗示FtMT2在苦荞氧化胁迫耐受性中发挥作用。(4)通过Genome walking技术克隆得到了2.1 kb的FtMT2启动子,顺式作用元件预测结果显示,该启动子区域脱落酸响应元件(abscisic acid responsive element,ABRE)、茉莉酸甲酯响应元件(methyl jasmonate responsive element,MeJa-RE)、热休克响应元件(heat stress responsive element,HSE)以及参与光响应的“ATCT-motif”、“TCT-motif”“GATA-motif”和“I-box”等。特别指出的是,该序列中存在3个金属响应元件相似序列(MRE-like sequence):“5′-TGCAG-3′”,该元件与动物MTs基因启动子上参与重金属响应的元件相似。(5)启动子活性分析结果显示,在无Cu2+处理条件下,不同截短体都具有启动子活性,暗示FtMT2基因具有一定的本底表达;在Cu2+处理条件下,不同截短体的启动子活性均显著上调,其中包含3个金属响应元件(MRE)的2.1 Kb截短体(FtMT2PR1)和2个MRE的1.6 Kb截短体(FtMT2PR2)启动子活性分别提高了6.38倍和6.15倍,含有1个MRE的1.4 Kb截短体(FtMT2PR3)启动子活性提高了3.01倍,而不含预测的金属响应元件的0.98 Kb截短体(FtMT2PR4)启动子活性也增加1.81倍。结果说明预测的金属响应元件可能存在剂量效应,并且在启动子序列中还存在其它重要的金属响应元件响应于Cu2+的处理。成功构建了1.6 kb启动子及MRE突变体-gus报告基因的植物表达载体,并筛选到了T1代阳性转基因拟南芥植株,为确定FtMT2的金属响应元件奠定了基础;同时为构建重金属响应的报告系统提供基础的元件。(6)成功构建pCAMBIA3301-FtMT2过表达载体,并筛选到阳性的拟南芥T2代转基因植株,为研究FtMT2在植物体内的生物学功能奠定了基础。以苦荞植株叶片为材料,采用农杆菌注射法进行GFP蛋白的瞬时表达,结果显示有较强的荧光信号。本实验首次通过实验证明,在荞麦上使用叶片注射的方法进行基因的瞬时表达是可行的,该研究为荞麦基因的瞬时表达研究提供一条新的途径。
[Abstract]:Metallothionein (metallothioneins, MTs) is a low molecular weight protein cysteine rich, play an important role in plant resistance to heavy metals. Buckwheat (Fagopyrum tataricum) rich in flavonoids, is an important medicinal and edible crops, which is barren, resistance and good resistance to heavy metal. But at present so far, there are still no reports about the heavy metal tolerance mechanism of tartary buckwheat and buckwheat metallothionein expression regulation and function. This study through cloning of buckwheat type II metallothionein gene sequence (FtMT2), constructed the over expression of FtMT2 in Escherichia coli, yeast and Arabidopsis, hydroxyl radical and determination of the recombinant protein removal ability FtMT2, in order to reveal the function of heavy metal tolerance in buckwheat; through the cloning of FtMT2 promoter sequences, predicted cis elements in the promoter region, constructing two different truncated promoter. Luciferase reporter vector, using the instantaneous response transformation of buckwheat mesophyll protoplast of FtMT2 promoter of Cu2+, in order to reveal the response model of.FtMT2 cloning of FtMT2 gene promoter activity and functional analysis of the promoter has a reference value for the establishment of Cu2+ pollution detection technology, and laid the foundation for the analysis of the intrinsic mechanism of buckwheat heavy metal resistance. The main results and conclusions are: (1) FtMT2 gene contains 3 exons and 2 introns and the intron boundaries conforms to the "5 '-GT' and 'AG-3'" rule.FtMT2 encoding region is 237 BP, encoding 78 amino acids; molecular weight prediction encoding protein is 7.885 kD and isoelectric point of 4.55. The protein sequence contains 14 Cys, containing C-C, C-X-C on the N- side, C-X-X-C motif and a highly conserved sequence, C- "MSCCGGNCGCGS" ends with 3 "C-X-C" motif.N- And the structure of C- terminal separated connection region of 42 amino acids, these are typical characteristics of plant type 2 metallothionein. Protein sequence analysis showed that the system developed recently and dicotyledonous legume Sesbania. (2) determination of growth curve and plate colony analysis showed that over expression of FtMT2 in Escherichia coli coli TOP10 and Saccharomyces cerevisiae respectively for Cu2+ and Cd2+ increased tolerance. Suggesting that FtMT2 play a role in buckwheat tolerance to Cu2+ and Cd2+. (3) the determination of hydroxyl radicals in FtMT2 recombinant protein removal ability showed that hydroxyl radical removal rate is 50% when the protein concentration was 46.5 g/m L. suggested that FtMT2 stress play the role of tolerance in buckwheat oxidation. (4) through the Genome walking technology cloned 2.1 KB FtMT2 promoter cis element prediction results showed that the promoter region of abscisic acid responsive elements (abscisic acid responsive e Lement, ABRE), methyl jasmonate (methyl jasmonate responsive element response element, MeJa-RE), heat shock response element (heat stress responsive element, HSE) and involved in the light response of "ATCT-motif", "TCT-motif" and "GATA-motif" and "I-box". In particular, there are 3 similar metal responsive element the sequence of (MRE-like sequence): "the 5 '-TGCAG-3'", the element and animal MTs gene promoter in heavy metal response elements. (5) promoter activity analysis showed that in the absence of Cu2+ treatment under the condition of different truncated has promoter activity, suggesting that FtMT2 gene has a certain the expression of Cu2+ in the bottom; processing conditions, different truncated promoter activity were significantly up-regulated, which contains 3 metal responsive element (MRE) 2.1 truncated Kb (FtMT2PR1) and 2 MRE 1.6 truncated Kb (FtMT2PR2) The promoter activity was increased by 6.38 times and 6.15 times, with 1.4 truncated Kb 1 MRE (FtMT2PR3) promoter activity was 3.01 times higher than that without prediction of metal responsive element 0.98 truncated Kb (FtMT2PR4) promoter activity increased 1.81 times. The results show that the prediction of metal response element there may be a dose effect, and also have the response processing element in response to Cu2+ other important metal in the promoter sequence. Successfully constructed 1.6 KB promoter and MRE mutant -gus gene plant expression vector and screened positive T1 generation of transgenic Arabidopsis plants, to determine the FtMT2 metal response element basis at the same time; provide the basis for constructing the system response to the report of heavy metal elements. (6) the successful construction of pCAMBIA3301-FtMT2 expression vector, and screened positive Arabidopsis T2 transgenic plants, for the study of FtMT2 in plants Laid the foundation for the in vivo biological function. In Tartary Buckwheat plants leaves as material, transient expression of GFP protein was evaluated by Agroinfiltration, showed a strong fluorescence signal. This is the first time through the experiments prove that the method used in the injection of buckwheat leaf gene expression is feasible. The research provides a a new way for the study of transient buckwheat gene expression.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X173;Q943.2

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