【作者】 段彬江；

【导师】 刘中来；

【作者基本信息】 华中师范大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 随着科技进步、工业发展以及人们生活水平的提高，环境问题日益加剧，其危害性可归为三类：致畸、致癌、致突变性，称为三致性，而三致性的根本又在于致突变性，致畸、致癌常常是致突变的结果。因此，检测环境中的各种具致突变性的因素以及物质一直是众多生物学者的研究热点。蚕豆微核技术即是一种较有效的致突变性检测手段之一。另外，由于环境对生物的负面影响，生物本身为了生存必须适应环境，在其长期的进化长河中逐步获得了一系列的适应机制来消除或部分减轻这些不良影响，而热激蛋白就是生物在受到环境胁迫时产生的并能帮助细胞免受胁迫伤害的一类蛋白质。本实验即主要以蚕豆为材料研究了重金属Cr⁶⁺与高温两种常见环境因素对蚕豆根尖细胞微核率的影响，以大麦为材料初步探讨了温度对植物叶片热激蛋白的诱导，并摸索了一套比较经济且方便的植物叶片可溶性蛋白双向电泳的方法。 1．环境对蚕豆根尖细胞微核率的影响 利用蚕豆根尖细胞微核技术比较研究了Cr⁶⁺和温度对植物根尖细胞微核率的影响，证实了温度和Cr⁶⁺等重金属一样，也可以对蚕豆根尖细胞的细胞核和染色体产生影响，其遗传学毒理效应与危害性也相似。当处理温度在28℃-34℃之间递增时，蚕豆根尖的微核率虽然变化不大，不过仍然呈现递增关系，而当温度在37℃-43℃递增，则呈现很明显的递增关系；但当温度升高到46℃以后，其微核率却反而降低。这与Cr⁶⁺和蚕豆根尖细胞微核率以及以往的相关实验结果都相吻合。 2．蚕豆叶片可溶性蛋白提取及双向电泳 以蚕豆叶片为材料，探讨了植物叶片可溶性蛋白在提取、双向电泳中的等电聚焦、平衡以及固定染色方面的一些改进，并通过银染得到了清晰的双向电泳图谱，从图中可肉眼观察到300多个清晰的蛋白斑点，等电点多在4-7之间，分子量在14-100kDa之间。⑨羔糕3.温度对大麦叶片热激蛋白的诱导 以大麦叶片为材料，利用355一Met作为蛋白标记，通过对双向电泳中等电聚焦后的凝胶PH值的测定，结合双向电泳图谱，简单地分析了双向电泳的分辨能力与等电聚焦后的凝胶中的PH梯度之间的关系;并通过SDS一PAGE着重研究了大麦叶片在黑暗和光照的条件下诱导热激蛋白强度的差异。结果表明，凝胶在PH值6一8.4区间，其pH值与PH值测定点间的距离成直线关系，形成了良好的pH梯度，所以此区间对蛋白质的分离效果最佳，分辨能力也最强。除此，本实验首次发现在相同的温度热激处理下，植物在黑暗条件下的热激反应比光照下要强烈，热激蛋白的合成量也更多一些，其机理尚待进一步研究。更多还原

【Abstract】 Following the progress of science and technology, the development of industry and the improvement of standard of people’ s living, the problems of environment prick up increasingly. The imperils can turn to three kinds: aberration, carcinogen and mutant. And the root of them rest with mutant, because both aberration and carcinogen often result from mutant. Thus, monitoring all kinds of mutant factors and materials in our environment is always the study hotspot by many biologists. Vicia micronucleus technology is just one of rather efficient measures of monitoring mutagens. In the other hand, because of the negative effects on the organisms by the surroundings, they themselves have to adapt to live. So they have tried to acquire a series of adaption mechanism to avoid or partly alleviate the bad effects step by step during the long evolutional period. The HSPs belong to these sorts of proteins which can be induced by heat shock and help avoiding the hurt from it. This experiment chiefly study on the comparative effects of micronucleus frequency on Vicia root tips after treating with two familiar factors: heat shock and Cr6+. Then by use of barley, discuss preliminaryly the HSPs induced by temperature and fish out a suit of quite economical and convenient method of two-dimensional gel electrophoresis of soluble proteins from the leaves of plant.1. Effects on micronucleus frequency of Vicia root tips induced by environmental factorsApplying with micronucleus test of Vicia root tips, the paper comparatively study on the effects on micronucleus frequency of Vicia root tips after treating with heat shock and Cr6+, and confirm temperaturecan affect the cell nucleolus and chromosome of Vicia root tips as heavy metal. And the genetic toxicological effects and imperils are also comparable. When the treating temperatures increase by degrees from 28癈 to 34℃, the micronucleus frequency of Vicia root tips increase though it is very slight. Also, between 37℃-43℃, the micronucleus frequency of Vicia root tips increase clearly. But when the temperature add up to 46℃, the micronucleus frenquency decrease on the contrary. Such are also tallied with the relative experimental results of Cr6+ and other heavy metals ago.2. Research on the isolation and two-dimensional gel electrophoresis of soluble proteins from the leaves of plantBy use of Vicia, this paper studies on soluble proteins of leaves of plant, including isolation, the course of two-dimensional gel electrophoresis, and dying. Following these courses, stable and rather clear map of two-dimensional gel electrophoresis is obtained through silver dying, and more than 300 spots can be observed by naked-eye. Most of their isoelectric points are from 4.0 to 7. 0, and their molecular weight are from 14 to lOOkDa.3. The HSPs of the leaves from barley induced by temperature Applying with the protein marker of 35S-Met, this paper study on thetesting of pH values in the gel after IEF of two-dimensional gel electrophoresis. Linking to the map of two-dimensional gel electrophoresis, then analyze simply the relation between resolving power of two-dimensional gel electrophoresis and pH values in the gel after IEF. Moreover, this paper stresses on the differences of intensity of HSPs induced by heat shock in the dark and in the light. The results indicate, the pH values between 6 to 8. 4 in the gel are linear with the distances between the testing spots, and form the well-grads of pH values. So the effect of proteins separation and the resolving power are thebest of all. Besides, this experiment finds at first plants response more intensively in the dark than in the light at the same temperature of heat shock, and gets more synthetical HSPs. But the mechanism need furthermore study.更多还原