多环芳烃胁迫下小麦根系质膜差异蛋白质组学研究
发布时间：2019-05-16 09:13
【摘要】：多环芳烃(Polycyclic Aromatic Hydrocarbons,简称PAHs)是一类致癌性很强的有机污染物。课题组前期研究证实了植物根系吸收PAHs的过程同时存在主动和被动运输两种转运方式,并揭示了植物根系的主动吸收是以H+/PAHs共转运的方式进行。关于菲胁迫下植物根系质膜蛋白差异组学的研究迄今尚未见有报道,而质膜蛋白质差异组学的研究对于阐释植物根系吸收PAHs的生物学机制至关重要。为此,本文首先以小麦为材料,探索根系质膜蛋白含量与影响根系生长的重要环境因素(温度、光照强度及培养时间)之间的关系,然后对SDS-PAGE凝胶浓度、等电聚焦时间、上样量等双向电泳条件进行优化,建立了小麦根系质膜蛋白的双向电泳技术条件;接着以菲(PHE)为多环芳烃代表,采用建立的双向电泳条件进行小麦根系菲胁迫下质膜蛋白质的差异表达分析,并对部分差异表达蛋白进行质谱鉴定,为多环芳烃质膜转运蛋白的分离鉴定以及PAHs污染环境的植物修复提供科学依据与技术支持。论文的主要结论如下:1、以小麦为材料,研究温度、光照强度和培养时间对根系生物量和质膜蛋白含量的影响。结果表明,当小麦在温度25℃、光照强度18 000 Lux、培养时间为3d时根系质膜蛋白质含量达到最高。正交试验结果进一步证实,25℃和18 000 Lux下培养3d,根系质膜蛋白含量最高,达3.7891 μμg/(10g),这一结果为质膜差异蛋白质组学的研究提供了培养方法。2、通过对小麦根系质膜蛋白双向电泳条件进行优化,确定了以450μg/gel的最佳蛋白上样量,10%的最佳SDS-PAGE凝胶浓度,18 h的最佳等电聚焦时间的双向电泳体系,得到了分辨率较高,重复性较好的质膜蛋白双向电泳图谱,并通过正交试验进一步验证了这一结果。3、以菲为多环芳烃代表,以菲处理4 h后和未经菲处理的小麦根系为试验材料,对两组试验材料的质膜蛋白质进行双向电泳分离,双向电泳图谱经过PDQuest软件分析,每组图谱均能得到200余个清晰的蛋白点;通过对两组图谱的比较,共得到了 14个显著差异表达的质膜蛋白;采用MALDI-TOF/TOFMS质谱系统对14个差异表达的蛋白进行一级质谱(PMF)和二级质谱(MS/MS)分析,并利用NCBI蛋白数据库检索质谱数据。被鉴定的差异蛋白包括白蛋白、钴氨素非依赖蛋氨酸合成酶、烯醇化酶、热休克蛋白、β-葡萄糖苷酶、甲胎蛋白、甘油醛-3-磷酸脱氢酶,这些蛋白分别参与了协同调控、信号传导、胁迫反应、物质运输和能量代谢等过程。综上试验结果,菲胁迫对小麦根系质膜蛋白的组成造成了很大的影响,并迫使根系质膜启动了多种应对菲胁迫的应激反应。
[Abstract]:Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons,) is a kind of organic pollutants with strong carcinogenicity. The previous studies of the research group confirmed that there were both active and passive transport modes in the process of PAHs uptake by plant roots, and revealed that the active absorption of plant roots was carried out by H / PAHs co-transport. Up to now, there are no reports on the differential histology of plasma membrane proteins in plant roots under phenanthrene stress, and the study of plasma membrane proteome is very important to explain the biological mechanism of PAHs uptake by plant roots. In this paper, wheat was used as material to explore the relationship between the content of plasma membrane protein and the important environmental factors (temperature, light intensity and culture time) affecting root growth, and then the concentration of SDS-PAGE gel and isoelectric focusing time. The two-dimensional electrophoresis conditions such as sample size and other two-dimensional electrophoresis conditions were optimized, and the technical conditions of two-dimensional electrophoresis of wheat root plasma membrane proteins were established. Then, using phenanthrene (PHE) as representative of polycyclic aromatic hydrocarbons (PAHs), the differential expression of plasma membrane proteins under phenanthrene stress in wheat roots was analyzed by two-dimensional electrophoresis, and some differentially expressed proteins were identified by mass spectrometry. It provides scientific basis and technical support for the isolation and identification of polycyclic aromatic hydrocarbon membrane transporter and the phytoremediation of PAHs pollution environment. The main conclusions of this paper are as follows: 1. The effects of temperature, light intensity and culture time on root biomass and plasma membrane protein content were studied. The results showed that the protein content of root plasma membrane reached the highest when the temperature was 25 鈩，
本文编号：2478176
[Abstract]:Polycyclic aromatic hydrocarbons (Polycyclic Aromatic Hydrocarbons,) is a kind of organic pollutants with strong carcinogenicity. The previous studies of the research group confirmed that there were both active and passive transport modes in the process of PAHs uptake by plant roots, and revealed that the active absorption of plant roots was carried out by H / PAHs co-transport. Up to now, there are no reports on the differential histology of plasma membrane proteins in plant roots under phenanthrene stress, and the study of plasma membrane proteome is very important to explain the biological mechanism of PAHs uptake by plant roots. In this paper, wheat was used as material to explore the relationship between the content of plasma membrane protein and the important environmental factors (temperature, light intensity and culture time) affecting root growth, and then the concentration of SDS-PAGE gel and isoelectric focusing time. The two-dimensional electrophoresis conditions such as sample size and other two-dimensional electrophoresis conditions were optimized, and the technical conditions of two-dimensional electrophoresis of wheat root plasma membrane proteins were established. Then, using phenanthrene (PHE) as representative of polycyclic aromatic hydrocarbons (PAHs), the differential expression of plasma membrane proteins under phenanthrene stress in wheat roots was analyzed by two-dimensional electrophoresis, and some differentially expressed proteins were identified by mass spectrometry. It provides scientific basis and technical support for the isolation and identification of polycyclic aromatic hydrocarbon membrane transporter and the phytoremediation of PAHs pollution environment. The main conclusions of this paper are as follows: 1. The effects of temperature, light intensity and culture time on root biomass and plasma membrane protein content were studied. The results showed that the protein content of root plasma membrane reached the highest when the temperature was 25 鈩，
本文编号：2478176
本文链接：https://www.wllwen.com/kejilunwen/huanjinggongchenglunwen/2478176.html
