【作者】 陈翠云；

【导师】 张立新；

【作者基本信息】 兰州大学 ， 植物学， 2008， 博士

【摘要】 由于EGY1是叶绿体中一个不依赖ATP的金属蛋白酶,推测它可能在光合作用功能发挥中具有一定的作用。本文通过反向遗传学的方法筛选获得拟南芥EGY1基因缺失的突变体（简称egy1突变体）。并采用转基因技术获得了互补苗,然后采用比色法、抗体制备、双向凝胶电泳、免疫共沉淀试验、RT-PCR、WesternBlot、Northern Blot及波谱技术等方法和手段对EGY1基因缺失影响光合作用功能与叶片衰老进行了系统研究,具体结果如下:1、与野生型相比,egy1突变体叶色黄绿,生长略微缓慢。随着叶片的发育,突变体的叶绿素含量、Fv/Fm以及光系统Ⅱ含量显著下降,表明egy1突变体的光合作用功能受到了影响。进一步通过蛋白免疫印迹发现光系统Ⅱ的反应中心蛋白D1、D2和外周天线蛋白LHCⅡ含量显著降低,而其它复合物蛋白与野生型没有区别,说明egy1突变体内主要是光系统Ⅱ受损。2、蓝色温和胶一向电泳和SDS-PAGE变性胶二向电泳之后免疫印迹检测发现EGY1的迁移位置与光系统Ⅱ的D2蛋白一致,而与光系统Ⅰ的PsaA/B蛋白不同,说明EGY1可能与光系统Ⅱ相结合;进一步His-EGY1和His-D2融合蛋白免疫共沉淀结果发现EGY1与D2共沉淀,说明EGY1确实与光系统Ⅱ相结合。3、光系统Ⅱ的含量降低主要有两种途径:合成减少和稳定性降低。本研究发现:egy1突变体与野生型相比,光系统Ⅱ的合成仅有10%的减少,而合成的略微减少不可能产生如此明显的光合作用功能受损,因此我们推测EGY1的缺失主要影响了光系统Ⅱ的稳定性。进一步通过影响稳定性的环境因子如冷、热、盐及光抑制处理进行了验证,结果显示:热、冷以及不同盐胁迫处理后,光系统Ⅱ的含量都有降低,尤其热胁迫降低最为显著,而作为对照的细胞色素b₆f复合物含量却没有变化,说明egy1突变体内,光系统Ⅱ不稳定从而对环境胁迫更敏感。1200μmol m^-2s^-1的高光强下(正常生长光强为120μmol m^-2 s^-1),egy1突变体内,光系统Ⅱ的D1和LHCⅡ蛋白含量随着光照时间的延长降低快于野生型,而PsaA/B、Cytf和CF1β蛋白都没有明显差异;30μmol m^-2 s^-1的弱光下,光系统Ⅱ的D1和LHCⅡ蛋白含量随着光照时间的增加突变体与野生型没有差异,表明光抑制处理下,egy1突变体内光系统Ⅱ更加不稳定更易被破坏。4、随着叶片发育,egy1突变体内衰老生理指标如叶片存活率和可溶性蛋白含量降低,而离子渗漏率则升高;同时,暗处理3天后,egy1突变体叶片的黄绿表型更加显著,暗示EGY1缺失可能导致叶片早衰;进一步通过核酸杂交分析检测基因转录水平的变化,发现突变体内衰老相关基因SAG12、SAG24和SEN4的转录水平有一定的升高,而光合相关基因CAB和RBCS的转录水平有所降低,说明egy1突变体的衰老进程明显较野生型快。egy1突变体中,幼叶的光合作用功能已经受损,但衰老还未起始,因而认为EGY1基因缺失使得叶片衰老加快是由光合作用功能受损所致。5、进一步对EGY1基因缺失导致叶片衰老的机理从己糖激酶信号途径（因为己糖激酶信号途径是糖类信号途径的主要组份）进行了研究,结果发现外源施加不同浓度的葡萄糖都对egy1突变体的胚轴伸长以及叶片转绿没有作用,说明突变体内的己糖激酶依赖型的糖类信号途径不受影响,也即egy1突变体的早衰与己糖激酶信号途径无关。总之,本论文首次揭示了拟南芥EGY1基因在光合作用功能及叶片衰老过程中所起的作用,并对这一基因缺失引起的光合作用功能受损及早衰机制进行了探讨,为叶绿体蛋白酶的研究现状增添了新的内容,也为深层次阐述植物光合作用功能及衰老的分子机制提供了理论基础和科学依据。更多还原

【Abstract】 Since EGY1 is a membrane-associated and ATP-independent metalloprotease that is localized in the chloroplast,it maybe plays roles in the function of photosynthesis.In the study,the homozygous egy1 mutants of Arabidopsis thaliana were obtained through reverse genetics analysis.The effects of EGY1-defect on function of photosynthesis and leaf senescence were studied by using colorimetry,antibody preparation,two-dimensional gel electrophoresis,pull-down assays,RT-PCR,Western Blot,Northern Blot,and spectrum-technique.The detailed results were summarized as follows:1.Compared to wild type,egy1 mutants were yellow-green and had a slight decrease in growth rate.In egy1 mutants,Fv/Fm,the chlorophyll and photosystemⅡ(PSⅡ) contents significantly decreased with increasing leaf age,suggesting that the function of photosynthesis was damaged.Immunoblot analyses further showed that the accelerated reductions in the abundance of D1,D2 and LHCⅡoccurred with leaf age in egy1 mutants.These results suggested that the EGY1 defection resulted in PSⅡdamage.2.Thylakoid membrane proteins were separated by BN-PAGE and SDS-PAGE,and then were used to immunoblot analyses.The results showed that the migration of EGY1 was consistent with that of PSⅡcomplex.Pull-down assays suggested that EGY1 was associated with PSⅡand colocalized with D2 protein.3.The synthesis of PSⅡwas almost unchanged in egy1 mutants(about 90%of wild type).It is impossible that the markedly impair function of photosynthesis was due to the slight decrease in PSⅡsynthesis.Thus,PSⅡstability was measured.It was reported that PSⅡstability was significantly reduced when egy1 mutants were treated by hot,cold and/or different salt stress.At the same time,PSⅡwas less stable under high light in egy1 mutants than in wild-type plants.These results suggested that the defection of EGY1 resulted in the loss of photosynthesis function through decreasing the PSⅡstability. 4.Leaf survival and soluble protein contents decreased whereas ion leakage of leaf increased significantly with increasing leaf age in egy1 mutants.Furthermore,the yellow-green phenotype of egy1 mutants was accelerated after darkness treatment for 3 days.These results suggested that the defection of EGY1 could induce the accelerated leaf senescence.Northern blotting analysis showed the transcript levels of senescence-associated genes(SAG12,SAG24 and SEN4)increased more and photosynthesis genes(CAB and RBCS)decreased more in egy1 mutants compared to wild-type plants,documenting the accelerated leaf senescence in egy1 mutants. According to the changes of photosynthesis function in young leaves of egy1 mutants, we speculated that the accelerated leaf senescence was caused by the damage of photosynthesis function.5.The egy1 seedlings didn’t show hyper-responsive phenotype to exogenously applied sugar,indicating that a hexokinase-dependent sugar-signalling pathway might not be affected by the loss of EGY1.That meaned the accelerated leaf senescence in egy1 mutants was hexokinase-independent sugar-signalling pathway or hormone -dependent signal pathways.In a word,the roles and detailed mechanisms of EGY1 on photosynthesis function and leaf senescence in Arabidopsis thaliana were studied for the first time.These results are helpful to people’s understand in chloroplast proteases,and provide us important information to comprehend the molecular mechanism of photosynthesis function and leaf senescence in plants and other systems.更多还原