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拟南芥ndr1突变体对UV-B辐射的抗性生理响应研究
郭琬莹
学位类型硕士
导师姚银安
2015
学位授予单位中国科学院大学
学位授予地点北京
学位专业生物工程
关键词拟南芥 Ndr1突变体 Uv-b辐射 抗逆 实时定量pcr
摘要在增强的UV-B辐射(ultraviolet-B radiation)作用下,植物体会受到一定的影响,产生活性氧自由基(ROS),造成DNA、蛋白质的损伤,使茉莉酸、水杨酸及乙烯等植物激素的含量发生变化。迄今为止,有研究发现植物在受到UV-B辐射时有两条通路在抵御紫外线伤害时发挥作用,一条是通过特异的紫外光受体UVR8来启动自身的应答机制来减弱辐射的伤害程度,另外一条是通过MAPK途径传导UV-B辐射,但是这两条通路的研究都还并不完善。本实验以模式植物拟南芥哥伦比亚(col)野生型、cim7(Constitutive immunity 7)突变体和ndr1(Non-race-specific disease resistance 1)突变体为研究材料,目的是揭示NDR1(Non-race-specific disease resistance 1)基因缺失时紫外线辐射对植物生理生化的影响及初步探讨紫外线辐射下植物的保护机制。 主要结果如下: (1)在增强UV-B辐射后,拟南芥野生型、ndr1突变体及cim7突变体的生长都受到了抑制。从增强UV-B辐射后三种拟南芥叶片的表型看出,辐射对叶片造成了不同程度的损伤, cim7叶片受到损伤最为严重,叶片明显萎蔫,叶色变为黄绿色;野生型其次,叶片萎蔫,叶色稍暗;ndr1突变体受到伤害相对较小。通过电子透镜的观察,ndr1内部叶绿体结构完整。进一步确认ndr1突变体的叶片对UV-B辐射的敏感性较弱,野生型和cim7突变体对UV-B的敏感性较强。 (2)在生理生化方面,增强UV-B辐射后,三种拟南芥受到了氧化胁迫,细胞内的活性氧物质过氧化氢的含量明显上升,丙二醛含量也明显升高,叶绿素含量均降低,其中ndr1突变体膜脂氧化程度较高。三种拟南芥的紫外吸收物质的含量有所上升。col野生型、ndr1突变体和cim7突变体细胞中的抗坏血酸和谷胱甘肽的含量都有所升高,其中ndr1突变体在处理前后SOD、POD活力较高,处理后的CAT和APX活力也明显高于另外两种拟南芥。 (3)通过Real time PCR分析,拟南芥中UVR8途径中的AtHY5基因的表达量有明显升高,同时,拟南芥细胞内的抗逆相关基因AtPR1、AtPAL2的表达量也有明显的上调。在ndr1突变体中,苯丙烷代谢相关的AtC4H基因以及和类黄酮合成相关的At4CL3和AtCHS基因在处理前表达量较高,AtPAL2基因在处理后表达量明显 升高。在ndr1突变体中,和DNA损伤修复相关的切除修复基因,如AtCSA、AtDDB1b和AtRAD54的表达量有明显的升高。在野生型和cim7突变体中,和DNA损伤修复相关的切除修复相关基因AtDDB2、AtUVH1和错配修复基因AtMSH2、AtMSH6的基因表达量都有上调。处理前后ndr1突变体的细胞周期相关的AtCYCLIN、AtHIS4基因的表达量较低。处理后的cim7突变体中AtCYCLIN基因有明显下调,野生型中的AtSIM基因有明显上调。 从上述实验结果得到结论:拟南芥ndr1突变体对于增强的UV-B辐射较野生型不敏感,NDR1基因的缺失对植物抵抗UV-B辐射来说有一定积极作用。; Ultraviolet-B(UV-B)radiation is a negative impact factor to plant growtrh. It cause the reactive oxygen species(ROS) burst in plant cell and damage to DNA and protein. Meanwhile, the contents of plant hormone such as jasmonic acid, salicylic acid and ethylene were changed by UV-B radiation. Up to now, two biological paths were found to against UV-B radiation stress in plant. One is to start their own response mechanism through specific receptors of UVR8 ultraviolet radiation to reduce the damage, and another is through the MAPK pathway to avoid the harmful from UV-B. But there did not have detail researchs focus on these two pathways. Using Arabidopsis thaliana wild-type col, mutant ndr1(Non-race-specific disease resistance 1) and mutant cim7(Constitutive immunity 7), we explored the physiology and biochemistry responses and the protection mechnisms of plant to strong UV-B radiation. The results were as follows: (1) Under strong UV-B radiation, the growth of wild-type, ndr1 and cim7 have been suppressed. Radiation caused varying degrees of damage on Arabidopsis thaliana leaves. cim7 was severely damaged, and leaves was turned to obvious wilt and yellow-green. leaves of col were also wilted and turned to dark. ndr1 were minor injured. By the transmission electron microscope, we found chloroplasts structure were intact in ndr1 plants. Its further confimerd that ndr1 were less sensitivity to UV-B radiation than col and cim7. (2) In physiological and biochemical aspects, three kinds of Arabidopsis thaliana faced oxidative stress under strong UV-B radiation, ROS substance such as hydrogen peroxide obviously increased, MDA was also increased, the chlorophyll content was decrease. The ndr1 mutant of Arabidopsis thaliana had a higher degree of membrane lipid oxidation. The ultraviolet absorption substances were increase. The content of ascorbic acid and glutathione were increase in col, ndr1 and cim7. ndr1 had a high activity of SOD and POD before and after treatment. The activity of CAT and APX were higher than others after treatment. (3) Using Real time PCR technique, gene expression data indicated that the expression of AtHY5 gene which is a key gene in the UVR8 pathway was up-regulated in all three Arabidopsis thaliana . Resistance related gene of Arabidopsis, such as AtPR1 and AtPAL2 was also up-regulated expression. In ndr1, phenylpropanes metabolizing gene AtC4H and flavonoid biosynthesis related geneAtcyclin genes At4CL3 and AtCHS were also expressed with high level before treatment, but AtPAL2 gene had a obviously increase after treatment. After treatment, DNA excision repair genes, like AtCSA, AtDDB1b and AtRAD54 were up-regulated in ndr1. DNA excision repair genes AtDDB2, AtUVH1, mismatch repair gene AtMSH2 and AtMSH6 were up-regulated. Cell cycle genes AtCYCLIN and AtHIS4 showed lower expression in ndr1 before and after treatment. After treatment, expression of AtCYCLIN was down-regulated in cim7, expression of AtSIM was up-regulated in col. In conclusions: ndr1 was not sensitive to strong UV-B radiation compared to wild-type, the deletion of NDR1 gene have certain positive effect in resistant to UV-B radiation.
学科领域生物工程
语种中文
文献类型学位论文
条目标识符http://ir.xjlas.org/handle/365004/14676
专题研究系统_荒漠环境研究室
作者单位中科院新疆生态与地理研究所
推荐引用方式
GB/T 7714
郭琬莹. 拟南芥ndr1突变体对UV-B辐射的抗性生理响应研究[D]. 北京. 中国科学院大学,2015.
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