He-Ne激光对小麦增强UV-B辐射损伤的修复效应及机理

【作者】 韩榕；

【导师】 王勋陵；

【作者基本信息】 西北大学 ， 植物学， 2002， 博士

【摘要】 本文采用He-Ne激光(5mW．mm^-2)辐照方法，对增强UV-B(10．08kJ．m^-2．d^-1)辐射下，小麦幼苗在生长、发育、生理、生化等方面的损伤修复效应，从个体、细胞、DNA分子等水平上进行了全面的研究和分析。主要阐明了He-Ne激光促进DNA分子损伤修复的途径、方式和机理。结果表明： （1）经增强UV-B辐射后，小麦明显表现出植株矮化、叶片卷曲、颜色加深，并产生“翘根”（胚根向上弯曲）现象。再以He-Ne激光处理后，小麦株高增加，侧根增多，“翘根”减轻，具明显的促进作用。从研究结果看，He-Ne激光辐照增强了UV-B辐射后小麦在萌发初期的淀粉酶活性、蛋白质和RNA的合成，从而促进了小麦的萌发与生长。 （2）He-Ne激光辐照后，促进了小麦种子的萌发能力。增强UV-B辐射组（B）与激光后处理组（BL）间的差异较显著（P<0．05）。激光对UV-B处理后小麦幼苗发芽势、发芽率的促进作用大于生长势，说明这种促进作用在萌发早期表现的较为明显。 （3）对染色体及细胞分裂的研究结果表明，增强UV-B辐射能抑制小麦细胞的有丝分裂率，产生落后染色体、染色体桥、游离染色体、核变形等畸变。其中，落后染色体和游离染色体较普遍，分别占总畸变率的32．8％和26．6％。并在UV-B诱导的小麦根尖细胞中，首次发现染色体在有丝分裂的后期到末期，分成3束、4束和6束等异常分裂新类型。本研究将之称为体细胞染色体的“多束分裂”或“分束分裂”。它们大体分布在两极，但两极上染色体“束”数有可能不同。同一“束”上染色体的数量也不完全相等。“束”之间未见有细胞壁的形成，因而，导致“多束体”的产生。 （4）采用扫描电镜对小麦叶表面结构观察的结果表明，He-Ne激光能使UV-B辐射后小麦叶表面蜡质的分布等级降低。 （5）通过小麦幼苗丙二醛（MDA）、谷胱甘肽（GSH）、抗坏血酸（AsA）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）和紫外吸收物含量及活性变化，测定了He-Ne激光在清除增强UV-B辐射导致的自由基方面的能力。He-Ne激光辐照可使UV-B辐射后小麦幼苗MDA的含量明显减少；AsA、GSH含量明显增加；SOD、CAT、POD等酶活性增加。说明He-Ne激光可通过酶促与非酶促抗氧化系统，增加小麦幼苗在生理水平对辐射损伤的修复能力。 （6）采用环丁烷嘧啶二聚体（Cyclobutyl Pyrimidine Dimers，CPDs）的特异性DNA内切酶——T4-Endo V（T4-核酸内切酶V）及琼脂糖凝胶电泳法，测定了He-Ne激光对由增强UV-B辐射引起的小麦细胞DNA中CPD切除的影响。与对照相比，5mW．mm^-2的He-Ne激光辐照，能降低由10．08kJ．m^-2．d^-1增强UV-B辐射小麦细胞DNA中形成的ESS（DNA中酶敏感位点——endonuclease sensitivesites）含量，亦即CPDs的数量。T4-Endo V酶切造成SSB数量的减少，即DNA中的酶敏感位点（ESS）含量的降低，说明He-Ne激光辐照促进了小麦细胞对CPDS的清除。 （7）用’H－TdR同位素标记V。麦DNA，通过种胚非按期DNA合成的变化，研究了He－Ne激光辐照对小麦DNA修复合成的影响。结果表明，增强UV习辐射导致DNA受损，从而诱发了细胞中非按期DNA的合成，并能通过DNA的修复合成，部分修复损伤的DNA。单纯He－Ne激光辐照处理可使小麦种子的S期提前，但S期的峰值与对照差别不大。He－Ne激光与UV－B复合处理可使非按期DNA合成期提前，修复合成速率提高。 （8）利用俱化乙锭（EB）只能插人双链 DNA中，并在激发光下显色的荧光光谱分析法，测定了He－Ne激光和增强UV－B不同处理后细胞中双链DNA （dsDNA）的含量，从而说明单链断裂的水平。在暗修复 sh时，各组 dsDNA含量差别达到最大。其中SL组降至最低，血＊＊A仅为0．39 p＊m卜；其次为B组①．423 fig·ml－l)和 BR组①．4：j pg·ml”＼ R、L组与对照差别不大，其 dsDNA含量变化也很小。BL组dsDNA含量的降低说明，He－Ne激光辐照明显促进了UV－B辐射后细胞中由于对DNA损伤链修复造成大量单链DNA 的形成 （SSDNA），而使得细胞中dSDNA的含量急剧减少。 （9）由于He－Ne激光辐照能够促进损伤细胞中CPD的减少，DNA修复合成及单链断裂数的增加，同时，与对照相比红光处理没有明显效应，因此认为：5mW·m”‘的HC－NC激光辐照能促进损伤DNA的修复，这种修复是一种暗修复，主要通过碱基和核苦酸切除修复来进行。 （l）通过与激光波长、功率相同的红光辐照对比研究，红光没有产生明显的效应。因此，可排除激光的光效应，且激光的热效应和压力效应也很小。那么，促进损伤修复的因子就是激光的磁场效应。对激光促进修复的机制分析认为：He．Ne激光通过其磁场效应作用于相应的多酶体系，从而表现出不同水平的损伤修复效应。 激光及UV－B辐射对生物体的效应已在多种植物中得到了广泛研究。但激光对增强UV－B辐射导致小麦在不同水平损伤的修复效应还未曾见有报道。本课题的研究为激光在生物体上的应用开辟了一个全新的领域，填补了国内在激光和UV－B辐射研究领域中的空白，居世界领先水平。它的研究无论在理论上，还是在实践中都具有重要的意义?更多还原

【Abstract】 The effects and mechanism of He-Ne laser (5 mW-mm"2) irradiation on the variation of morphs and structure, physiological metabolic activities and the DMA repair capacity in damaged wheat seedlings induced by enhanced ultraviolet-B (10.08 kJ-m"2-d~1) radiation were studied. The pathway and mechanism of the DNA repair stimulated by He-Ne laser irradiation were indicated chiefly.1) Under the enhanced ultraviolet-B radiation, the wheat seedlings shown the decreased height and leaf area, accumulation of screening pigments and ’root-bending’. But following He-Ne laser irradiation, the height and number of the branch root were increased, and the ’root-bending’ assay was mitigated. The activity of amylase and synthesis capacity of RNA and soluble protein were enhanced by He-Ne laser irradiation. This was the reason of increasing the wheat seedlings germination.2) After UV-B radiation the germination ability of the wheat was enhanced by He-Ne laser irradiation. Using t-test at P^O.05, there was significant difference between group of UV-B treated only and compound treatment group (with UV-B and He-Ne laser). The stimulation of the He-Ne laser irradiation effect on the vigor was clearer than the growth potential of germination and germination capacity. It indicated that the stimulation effect was expressed in early phase clearly.3) The results of studying on the chromosomal mutation and mitosis of the wheat irradiated by UV-B indicated that the rate of mitosis was inhabited by UV-B radiation (10.08 kJ ?m"2 ?d"1) in wheat cells, and some types of chromosome aberration were induced. Among of them, the fragments and lagging chromosomes were more than the correct, the rate of the fragments was 26.6%, lagging chromosomes was 32.8%. Under the condition of the enhanced ultraviolet-B radiation, the phenomenon of abnormal mitosis was first discovered, such as the three-bundle> four-bundle and six-bundle division of the chromosome when the cell is in anaphase. We think that the abnormal mitosis should be called ’multi-bundle division’ or ’partition-bundle division’ of the somatic chromosome. These ’bundles’ of the chromosomes were distributed two polar, but the number of the ’bundle’ is unequal possible between the two polar. In each ’bundle’, the number of the chromosome is different from another ’bundle’. There are not the cell walls be formed between in two bundles, so it will be form the ’poly-bundle bodies’.4) The results of epicuticular observed with the scanning electronmicroscope (SEM, KYKY-2800, China) shows that the wax distribution could be changed by He-Ne laser irradiation.5) Changes in the concentration of malondialdehyde (MDA), glutathione (GSH), ascorbic acid (AsA), and activity of superoxide dismatase (SOD), catalase (CAT), peroxidase (POD), and UV-B absorbing compounds (absorbance at 300 nm) were measured to test the capacity of He-Ne laser irradiation on clearing the free radical induced UV-B radiation.The damage of wheat seedlings induced by enhanced UV-B radiation in physiology can be repaired partly by He-Ne laser irradiation with enzymatic and non-enzymatic system including of decreasing the MDA concentration, increasing the contents of AsA, GSH, UV-B absorbing compound, and the activities of SOD, CAT, POD.6) Using agarose gel electrophoresis and T4-endodeoxyribonuclease-V, which is a kind of restriction endonuclease of cyclobutyl pyrimidine dimer (CPD), the impacts of He-Ne laser (5 mW-mm"2) irradiation on DNA excision repair capacity in damaged wheat cells induced by enhanced ultraviolet-B (10.08 kJ-m~2-d~1) radiation were studied. The results indicated that the content of endonuclease sensitive sites (ESS) was reduced by He-Ne laser irradiation, which formed in cells irradiated by enhanced ultraviolet-B. With the irradiation of He-Ne laser, the excision of CPDs and the reduction of single strand breaks (SSB) contents which were the endonuclease sensitive sites (ESS) digested by T4-endodeoxyribonuclease-V had been stimulated in the wheat cells.7) By更多还原