棉花幼苗叶片中谷胱甘肽转移酶响应盐胁迫的分析

发布时间：2018-02-25 23:16

本文关键词： 棉花谷胱甘肽转移酶蛋白质印迹法生物信息学盐胁迫　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：土地盐碱化作为一种非生物胁迫,已成为制约农业生产发展的重要限制因素。棉花作为世界上重要的经济作物,对高温、干旱、高盐等非生物胁迫具有一定耐性,但盐浓度过高会造成棉花渗透胁迫和离子毒害,导致棉花产量和质量的下降。谷胱甘肽转移酶(Glutathione transferases,GSTs,EC 2.5.1.18)是一类多功能蛋白家族,在植物体抵御外源物质毒害及抵抗多种非生物胁迫中起着重要作用。目前,对GST在耐盐方面的研究多集中在烟草、拟南芥等模式生物中,但是对棉花中GST的耐盐机理研究还鲜有报道。本研究首先利用生物信息学方法,对棉花谷胱甘肽转移酶理化性质、二级结构、信号肽及亚细胞定位、三维结构进行预测,对氨基酸序列进行了同源性比对并构建系统进化树。在此基础上以中棉所79为实验材料,利用NaCl溶液模拟盐胁迫对棉花叶片中GST的表达进行分析。对基于SDS-PAGE的western blot检测方法进行了优化,确定了适合western blot的最佳蛋白样品制备方法和电泳上样量,对盐胁迫下棉花GST的表达变化进行了分析。在此基础上,建立了基于双向电泳的western blot分析方法,得到了盐胁迫下棉花GST的差异表达图谱,并对western blot检测出的GST差异蛋白点进行量化分析,获得棉花GST亚型响应盐胁迫的表达情况。主要研究结果如下:1、对棉花中谷胱甘肽转移酶进行生物信息学分析。利用多种工具在理化性质、二级结构、亚细胞定位、亲水性/疏水性方面进行预测。结果显示GST含有225个氨基酸,分子量为26KDa,等电点为5.75,属于亲水性蛋白质,蛋白性质稳定为细胞质蛋白。利用Blastp进行同原序列比对,选择6条氨基酸序列构建系统进化树。2、优化了棉花叶片蛋白的制备方法并确定适用于western blot的上样量。在参考本实验室已有的提取方法及查找文献的基础上,筛选适合于1D-western blot检测GST的最适样品制备方法,最终确定了提取蛋白粉溶解上样的方法。通过不同上样量的western blot显影效果,发现GST适合于较高的上样量。3、利用SDS-PAGE和western blot方法对盐胁迫下的棉花谷胱甘肽转移酶表达量的变化进行分析。在盐浓度相同的条件下,不同的胁迫时间造成棉花叶片中GST的差异表达。结果显示,对照组棉花叶片内GST表达稳定,盐胁迫12小时后GST的表达受到抑制,处理24小时GST的表达量开始上升,并随着时间的增长而不断上升。4、建立了基于双向电泳技术的western blot检测体系。对胁迫24h的棉花幼苗叶片蛋白进行双向电泳分析,获得棉花叶片响应盐胁迫的差异蛋白图谱。在此基础上对蛋白凝胶经行western blot分析得到2D-western blot的差异蛋白点,利用ImageMaster 2D PLatinum7软件对不同的GST蛋白点进行匹配和量化分析。结果显示,棉花中共有8个GST蛋白点响应盐胁迫,并出现差异表达,其中2个点表达上调,6个点表达下调。这表明,棉花叶片中存在多种亚型的谷胱甘肽转移酶,且在盐胁迫处理后出现不同的表达情况,这一结果为后续针对谷胱甘肽转移酶亚型在耐盐机制中的作用及基因改良育种方面的研究打下基础。
[Abstract]:Land salinization as an abiotic stress, has become an important restriction factor for the development of agricultural production. Cotton as the most important economic crops in the world, the high temperature, drought, high salt and other abiotic stress has certain tolerance, but the salt concentration is too high will cause osmotic stress and ion toxicity of cotton, resulting in decreased yield and quality cotton. Glutathione S-transferase (Glutathione, transferases, GSTs, EC, 2.5.1.18) is a multifunctional protein family, resist exogenous substances and a variety of non poison resistance plays an important role in abiotic stress in plants. At present, the research of GST in salt area concentrated in tobacco, Arabidopsis and other model organisms. But the cotton GST in the mechanism of salt tolerance research is rarely reported. In this study, using bioinformatics methods, physical and chemical properties of cotton enzyme glutathione, two level structure, signal peptide and subcellular set A 3D structure prediction of the amino acid sequence homology and phylogenetic tree was constructed. On the basis of CCRI 79 as experimental materials, simulation of salt stress on the expression of GST in cotton leaves were analyzed by using NaCl solution. The Western blot detection method based on SDS-PAGE were optimized to determine the best protein samples preparation method and electrophoresis for Western blot sample, on expression of GST of cotton under salt stress were analyzed. On this basis, the two dimensional electrophoresis analysis of Western blot based on the difference, under salt stress of cotton GST expression profile, and the GST difference detection of Western blot protein. The quantitative analysis of cotton GST subtype expression in response to salt stress. The main results are as follows: 1, the glutathione S-transferase bioinformatics analysis using cotton. A tool in the physical and chemical properties, two level structure, subcellular localization, hydrophilic / hydrophobic side prediction. The results showed that GST containing 225 amino acids, molecular weight of 26KDa and isoelectric point of 5.75, is a hydrophilic protein, protein stability for cytoplasmic protein. Using Blastp in the same primary sequence, selection 6 amino acid sequence phylogenetic tree of.2 protein in cotton leaves, optimizing preparation method and determined for Western blot sample. Based on the existing laboratory reference extraction method and search literature, screening for 1D-western blot detection GST the optimum sample preparation methods, and ultimately determine the method extraction of protein powder dissolved sample. Through the Western blot imaging effect of different sample volume, sample volume of.3 found that GST is suitable for high, using SDS-PAGE and Western blot method under Salt Stress on cotton glutathiones Shift enzyme expression analysis. In the condition of the same concentration of salt under different stress time, differential expression in cotton leaves caused by GST. The results showed that the control group GST was stably expressed in cotton leaves, the expression of GST was inhibited by salt stress after 12 hours, the expression of 24 hours GST began to rise, and with the increase of time and the increasing of.4, established the Western blot detection system based on two-dimensional electrophoresis. For two-dimensional electrophoresis analysis of stress 24h of cotton seedling leaf protein, cotton leaf protein profile obtained in response to salt stress. On the basis of protein gel by Western blot analysis 2D-western blot proteins, matching and quantitative analysis of different GST protein using ImageMaster 2D PLatinum7 software. The results showed that a total of 8 cotton GST protein point in response to salt stress, and the difference The expression, of which 2 spots up-regulated and 6 spots were down regulated. This suggests that multiple isoforms of glutathione S-transferase in cotton leaves, and the expression in different salt stress treatment, the results lay the foundation for the further research on glutathione S-transferase isoforms in salt tolerance mechanism and genetically modified breeding.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S562

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