【作者】 郭炜；

【导师】 王敏琴；

【作者基本信息】 山东大学 ， 细胞遗传学， 2008， 硕士

【摘要】 彗星实验（comet assay）又称单细胞凝胶电泳（single cell gel electrophoresis,SCGE）,以电泳后细胞核DNA显示的显微镜荧光图像特征而得名,是用来定量分析和测定细胞核中DNA损伤程度的实验方法。可在单细胞水平上检测单链/双链DNA断裂,DNA分子中碱性不稳定位点,DNA链中不完全切除修复位点和DNA分子内及分子间交联等损伤与修复的的方法,具有敏感、稳定、快速的优点。在国外已得到了广泛应用,在我国SCGE主要被用于对环境毒理学、离子射线损伤、自由基损伤、癌症放化疗、生物监测等领域。对植物的研究报道甚少。本实验设计是建立在前期所做的植物不对称体细胞杂交实验基础之上。实验发现柴胡、盐芥的原生质体分别经UV（380μW/cm²）照射2min,5min后再分别与不同植物的原生质体融合,在所得的相应的体细胞杂种细胞中观察到柴胡和盐芥具有使异源染色质消减与渐渗的现象。该结果与本实验室所作的大量有关小麦的体细胞杂交实验和国内外其它已报道的其它植物体细胞杂交实验的结果相反。为了进一步探讨柴胡、盐芥为什么可使受体染色质消减与渐渗,本实验拟以六种不同植物的原生质体（单、双子叶植物各三种）为研究对象,对经UV辐照后导致细胞中DNA损伤测定的SCGE方法进行不同实验条件优化;利用CASP分析软件（comet assay software projiect）对所得数据进行分析;综合评价不同UV照射剂量下各植物DNA损伤程度的差异;对UV辐照前后与DNA损伤修复相关的抗氧化酶及生物大分子化合物的变化情况进行测定分析。选取的双子叶植物:柴胡（Bupleurm scorzonerifolium Willd.）,盐芥（Thellungiella salsuginea）,拟南芥（Arabidopsis thaliana）。单子叶植物:小麦（Triticum aestivum L.）,中间偃麦草（Thinopyrum intermedium（Hest）Nevski）,簇毛麦（Dasypyrum villosum）。以上研究期望对植物不对称体细胞杂交机理的阐明提供新依据。主要研究内容及实验结果:1)利用CASP软件对SCGE测定的细胞损伤量（Olive Tail Moment,OTM值）进行分析彗星实验测定六种待测实验材料的原生质体,在低剂量（380μW/cm²,≤2min）的UV辐照下,柴胡、盐芥、拟南芥、簇毛麦及小麦细胞的OTM值差别不明显,肉眼可辨其彗星电泳图片拖尾较短;中间偃麦草细胞的OTM值在UV照射1min剂量时,较其它5种材料上升明显,此时彗星电泳图片显示有较明显的拖尾现象。以中等剂量的UV（380μW/cm²,2-5min）辐照时,上述六种材料的OTM值差异显著,其中柴胡、盐芥、拟南芥细胞的OTM值比小麦、中间偃麦草、簇毛麦明显偏低,此时小麦、中间偃麦草、簇毛麦彗星电泳图片有较明显的拖尾出现;当UV照射剂量达到（380μW/cm²,5-10min）高剂量时,盐芥、拟南芥细胞的OTM值急剧上升与小麦、中间偃麦草、簇毛麦相近,此时柴胡细胞也有上升,但是OTM随UV照射剂量变化的曲线较其它三种细胞相对缓慢。在极端UV照射剂量下（380μW/cm²,12.5min）,除了柴胡其它5种材料细胞中的DNA在凝胶中都呈弥散状态已无法检测,而柴胡中还有接近20%细胞的DNA损伤可以被检测到。不同UV照射剂量对六种植物材料DNA损伤的比较图析显示在每个照射剂量下,柴胡、盐芥的OTM值都是偏低的,而簇毛麦、中间偃麦草的OTM值则是偏高的。以SCGE结合CASP软件分析可见六种植物细胞UV抗性由高到低的排列顺序是:柴胡＞盐芥＞小麦＞拟南芥＞中间偃麦草＞簇毛麦。2)UV照射前后各实验材料愈伤组织中O₂^-的含量测定未经UV辐照的六种材料细胞内都含有一定浓度的O₂^-,盐芥、拟南芥、中间偃麦草、簇毛麦细胞O₂^-的含量高于柴胡、小麦。随着UV照射剂量的增加,各实验材料细胞内O₂^-含量都有所上升。盐芥细胞内O₂^-浓度急剧上升;柴胡细胞内O₂^-上升较为平缓;其余四种材料在每个UV照射剂量下细胞内O₂^-含量处于柴胡与盐芥之间。推测柴胡细胞内或许存在与其它材料不同的,更为有效的清除UV胁迫产生O₂^-的机制。3)UV照射前后各实验材料愈伤组织中黄酮含量的比较利用紫外分光光度计进行UV（380μW/cm²,0,5,10,15,20min）照射前后六种实验材料愈伤组织中总黄酮含量测定时发现:UV照射前柴胡细胞中总黄酮的含量最高,小麦细胞中总黄酮的含量次之;中间偃麦草、簇毛麦细胞中总黄酮含量居中,盐芥、拟南芥细胞中总黄酮的含量最低。UV辐照5min内,柴胡、小麦细胞内总黄酮的含量与UV照射时间呈正相关,达到最高,然后开始降低:UV辐照10min内,盐芥、拟南芥细胞内总黄酮含量与UV照射时间呈正相关,此时测定的拟南芥、中间偃麦草、簇毛麦细胞中总黄酮含量达到最高,然后开始降低;盐芥细胞中总黄酮含量则在UV辐照15min后达到最高,之后又降低。4)UV照射前后各实验材料愈伤组织中多酚类化合物含量的比较紫外分光光度计测定UV（380μW/cm²,0,5,10,15,20min）照射前后各实验材料愈伤组织中多酚类物质含量显示:UV照射前后柴胡细胞中酚类物质含量在六种材料中最高,在一定剂量范围内UV（380μW/cm²,≤10min）两者呈正相关,UV辐照5min时,柴胡细胞中多酚类化合物含量达到最高;拟南芥、小麦、中间偃麦草、簇毛麦、盐芥细胞多酚类物质含量则在UV辐照10min时达到最高。通过黄酮、多酚类物质的含量对比,推测各种植物是分别通过不同的方式抵御其氧化造成的损伤。如柴胡、小麦细胞可能主要通过快速的应激反应产生大量的保护类物质,如黄酮、多酚类物质直接吸收UV已抵御其氧化造成的损伤,以防止细胞中的DNA遭受损伤;而拟南芥、中间偃麦草和簇毛麦细胞中黄酮、多酚类物质产生较慢,它们可能主要通过体内存在的抗氧化酶类直接清除UV氧化产生的氧自由基而起作用;而对照组盐芥细胞中黄酮类化合物几乎测不到,多酚类物质含量也很低,但UV照射后两物质含量迅速增加,推测细胞内游离的多酚类/黄酮类化合物与抗氧化酶类在清除自由基中相结合共同发挥作用。5)UV照射前后各实验材料愈伤组织中POD活性分析未经UV照射时,柴胡细胞中POD活性低于其它五种实验材料。随UV照射时间增加,各实验材料细胞中POD活性均呈现先升后降的振荡趋势。UV-5min处柴胡细胞中的POD活性上升仅次于POD活性最高的拟南芥;UV-10min时柴胡细胞中POD活性下降的速度大于其它五种,降至最低;但到UV-20min处柴胡细胞中POD活性与小麦、盐芥、簇毛麦相近但都低于拟南芥、中间偃麦草。说明轻度UV辐照可能会在一定程度上诱导POD活性,但高剂量UV辐照,会使POD活性有所下降。六种实验材料POD活性随UV变化总体未呈现统一的规律性。6)UV照射前后各实验材料愈伤组织中Pro浓度测定未经UV辐照时,拟南芥细胞内Pro含量最高,而盐芥的最低,其余材料的细胞中Pro含量居中,随着UV照射剂量的增加,六种材料细胞内Pro含量都有所上升。柴胡细胞内Pro浓度相对于其它五种细胞上升较缓慢,含量一直处于最低;盐芥细胞内Pro含量上升最快。当UV照射剂量大于10min,盐芥细胞内的Pro浓度高于其它五种细胞。推测盐芥细胞可能部分通过Pro的浓度变化来起到调节其抗UV氧化的作用。更多还原

【Abstract】 Comet assay as also called the single cell gel electrophoresis（SCGE）is a sensitive,reliable,and rapid method which can detect double or single DNA breaks and alkal-labile sites in eukaryotic individual cells.In this study,this method was applied to measure DNA damage of several plant protoplasts,which induced by UV. An improvement method that is used for detecting different plant DNA damages.In our previous asymmetric somatic hybridization of plants,some asymmetry hybrids were obtained by UV irridiated protoplasts of Bupleurm scorzonerifolium （380μW/cm²,5min）and Thellungiella salsuginea（380μW/cm²,2min）before asymmetric somatic hybridization between Arabidopsis thaliana/B,scorzonerifolium and A.thaliana/T,salsuginea.The chromotins of receptor eliminated and introgressed into genomes of donors were investgated in these somatic hybrids.On the contrary., many results showed that the chromosomes of donor fragmented,translocted or introgressed into the genomes of receptor in the somatic hybridization in which Triticum aestivum as one partners and heterologous genus grasses were UV-irradiated before fusion.Since ionization and excitation could be induced by primary UV-radiation led to the production of new chemical species in donor and hybrid cell, such as hydroxyl radicals,hydrogen radicals,and solvated electrons produced have a finite probability of interacting with DNA sites.To investigate the relationship between the dosages of UV-radiation on the protoplasts of donor and the chromosomes elimination/introgression of receptors or donors in the somatic hybrids. we use SCGE to evaluate UV damage at DNA level on six plant protopasts（B. scorzonerifolium,T.salsuginea,T.aestivum L.,Hasypyrum villosum and Thinopyrum intermedium（Hest）Nevski.）and using CASP（Comet assay software project） software to accurately analyze the images.The related physiological indicators produced by UV- radiation on plant materials were also detected and analyzed.The following experiments were operated in this research:The values of OTM（value of damage on cells）could reflect the response for different dosages of UV-irradiation on six plant protoplasts.We found that the value of OTM of B.scorzonerifolium,A.thaliana,T.salsuginea,T.aestivum and S,villosum don’t have obvious images of DNA smearing（DNA migration length）at the low doses of UV-irradiation.While the value of OTM of H.villosum is the highest in these plant materials.The images of SCGE had the longest DNA smearing after UV irradiated its protoplasts for 1 min.The OTM values of the other five protoplastes varied greatly during the medium doses of UV（380μW/cm²）irradiatied for 2-5min. The OTM values of B.scorzonerifolium and T.salsuginea are lower than that A.thaliana,T.aestivum and T.intermedium,accordingly,the images of SCGE have longer DNA smearing in A.thaliana,7.aestivum and 7.intermedium as compared to B.scorzonerifolium and T.salsuginea with increase in UV exposure time.The DNA damage level of 7.salsuginea rises sharply with increase in UV exposure time （380μW/cm² for 5-10min）using SCGE analysis;while the OTM values of B. scorzonerifolium rises gently.Just about 20%of DNA damages can be detected in the cells of B.scorzonerifloium,the other DNA samples all diffused in the gel to be detected with UV exposure time rised to（380μW/cm²）12.5min.According to above data,the OTM value of B.scorzoneriflium is the lowest and H,villosum,is the highest, respectively.UV-resistant order of six plant materials arranged descending roughly:B. scorzonerifolium＞T.salsuginea＞T.aestivum＞A.thaliana＞T.intermedium＞H. villosum.1)The concentration of flavonoids in all six kinds of plant cells was measured using AI（NO3）3-NaNO2-NaOH method before and after UV-irradiation.Before and after UV irradiation,the concentration of flavonoids in B.scorzonerifolium was the highest. and the concentration of flavonoids in A.thaliana and T.salsuginea are the lowestin all other plant materials mensurated by ultraviolet spectroscopy.Accompaned by the rise of UV dosage,the content of flavonoids in all six kinds of plant materials are all gradually increased and then decreased after reaching a peak.The concentration of flavonoids in B.scorzonerifolium cells reached the maximum at UV-5min,while in T. salsuginea cells reached the maximum at UV-15min.The concentration of flavonoids in A.thaliana,T.intermedium and H,villosum reached the peak at UV-10min.2)The concentration of phenolic compounds in B.scorzonerifolium,T.salsuginea, A.thaliana,T.intermedium,H.villosum and T.aestivum were measured after UV-irradiation using Prussian Blue（PB）method as compared to untreat sample.The concentration of polyphenols in B.scorzonerifolium is the highest in six plant materials.A sharp increase in the level of polyphenols was observed in six kinds of plant materials with increase in the time of exposure of samples to UV-radiation.A significant change in the content of polyphenols was observed in calli of B.scorzonerifolium till it reached the maximum level when added the UV irradiation for 5 min,and the production of polyphenols enhanced with increase the UV exposure for 10 min in all other five samples.Above results lead us to believe that UV radiation induced secondary metabolites accumulation have an important role in protection B.scorzonerifolium cells against UV radiation promoted flavonoids paths processes.3)The activities of POD in B.scorzonerifolium,T.salsuginea,Arabidopsis thaliana, H.villosum,T.intermediumand T.aestivum were detected before and after UV-irradiation.No significant regularity of POD content was observed in all six samples.Compared to other samples,the activity of POD in B.scorzonerifolium calli was the lowest in the control.Calli of B.scorzonerifolium had higher potential to accumulate POD in response to UV irradiation for 5 min,and then decreased sharply to the lowest level.Almost the same level of POD activity of B.scorzonerifolium. T.aestivum,T.salsugmea,and H.villosum was detected in the time of exposure of samples to UV-radiation for 20 min.The results suggested that UV could induce the activity of POD in plants. 4)The concentration of Pro in B.scorzonerifolium,T.salsuginea,A.thaliana,H. villosum,T.intermedium and T.aestivum were measured before and after UV exposure by acidic-ninhydrin method.The content of Pro was the highest in A. thaliana,and was the lowest in T.salsuginea in control samples.Calli of six kinds of sample exposed to UV radiation showed a time dependent increase in the level of Proline.No significant Pro accumulation was show in the calli of B.scorzonerifolium with increase in duration of UV exposure in its all five samples.Calli of T.salsuginea had higher potential to accumulate Pro in response to UV irradiation more than 10 min.Besides Proline accumulation in T.salsuginea during UV-exposure might be one of the modes to counteract UV radiation promoted ROS generation.5)The activities of O₂^?in B.scorzonerifolium,T.salsuginea,A.thaliana,H. villosum,T.intermedium and T.aestivum were measured before and after UV-irradiation by hydroxylammoniumchloride autoxidation method.The activities of O₂^?in the calli of B.scorzonerifolium and T.aestivum were the lowest in the six plants in controls.A sharp increase in the level of O₂^?was observed in T.salsuginea upon exposure to UV-radiation,and no significant change was observed in calli of B.scorzonerifolium.O₂^?generation is one of the initial cytochemical responses of plants exposed to UV radiation.To sum up,Secondary metabolite,such as flavonoids and polyphenols accumulation is the most frequent metabolic responses exhibited by B.scorzonerifolium exposed to UV stresses.POD and Pro accumulation in other plant samples during UV-exposure might be one of the modes to counteract UV radiation promoted ROS generation.更多还原