NO和钌红对桃果实线粒体MPTP和mtDNA拷贝数的影响

发布时间：2018-03-13 07:03

  本文选题：线粒体　切入点：一氧化氮　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：线粒体除了作为细胞中有氧呼吸和能量制造场所,还能够参与细胞中电子传递、细胞凋亡、细胞代谢等过程,在机体正常生理活动中发挥重要作用。一氧化氮(nitric oxide,NO)是一种具有脂溶性的生物活性物质,在生命信息传递中作为第一信使参与到植物体中多种生理反应。适量浓度的NO还可以通过调控线粒体的生理功能来影响植物幼苗的生长、果实的成熟和贮藏等过程。线粒体通透性转换孔(mitochondrial permeability transition pore,MPTP)控制不同离子进出线粒体,参与到细胞凋亡过程中,但具体的作用机理并不明确。线粒体DNA(mtDNA)由于自身结构的特点而易受到损伤,造成mt DNA的突变和缺失,成为细胞凋亡的控制因素之一。Ca~(2+)作为生物体中第二信使,也是生物体中重要的组成成分。钌红(RR)能够抑制线粒体中钙离子通道。探明NO和RR对mtDNA拷贝数的影响和对MPTP开放的影响,有助于探究NO对肥城桃桃果实采后贮藏的具体机理,对增加桃果实贮藏时间有重要意义。本实验以肥城桃桃果实为实验材料。从美国国家生物技术信息中心(National Center for Biotechnology Information,NCBI)获取的NAD1基因设计引物,以不同温度和不同处理下的桃果实总DNA为模板,通过实时荧光定量PCR和荧光显微镜来观察mtDNA拷贝数的变化情况。选取冷害最严重的5℃下的桃果实,用15μmol L~(-1) NO、5μmol L~(-1)c-PTIO单独处理和与20μmol L~(-1)钌红混合处理,测量线粒体中ROS含量、抗氧化酶活性、线粒体膜电位、线粒体通透性等指标,来研究NO对线粒体MPTP的影响。成功得到长度为159bp的目的基因片段。从NAD1基因的实时荧光定量PCR和荧光显微镜的结果中可以看出在0℃和5℃下,外源15μmol L~(-1) NO能够促进桃果实线粒体拷贝数的减小,而在常温下贮藏桃果实中,外源15μmol L~(-1) NO抑制了线粒体拷贝数的减小。钌红处理使0℃和5℃下处理的桃果实中mtDNA拷贝数得到增加。在线粒体膜通透性研究中,外源的15μmol L~(-1) NO处理能够减少桃果实线粒体中ROS含量而且使线粒体中抗氧化酶(CAT、SOD、POD)的活性得到提高;NO处理还能够使线粒体的耗氧量下降,进而抑制细胞的呼吸作用;NO处理一定程度上抑制了线粒体膜电位的降低,维持膜电位在高水平,同时提高了线粒体膜抗性,抑制线粒体膜透性的增加。15μmol L~(-1) NO-20μmol L~(-1) RR处理在短时间内降低了线粒体中ROS的含量,同时提高了抗氧化酶CAT的活性、抑制膜电势降低和膜透通性的开放。所以15μmol L~(-1) NO-20μmol L~(-1) RR处理可以在短时间抑制线粒体MPTP的开放。结果表明外源NO能够抑制线粒体MPTP的开放,维持线粒体内环境的稳定,减缓了桃果实中细胞凋亡的过程,使肥城桃桃果实采后贮藏时间得到增加。
[Abstract]:Mitochondria not only act as a place for aerobic respiration and energy production in cells, but also participate in the processes of electron transfer, cell apoptosis, cell metabolism, and so on. Nitric oxide (no) is a kind of liposoluble bioactive substance. As the first messenger in the transmission of life information, it is involved in many physiological reactions in plants. The proper concentration of no can also affect the growth of plant seedlings by regulating the physiological function of mitochondria. The process of fruit maturation and storage. Mitochondrial permeability transition transition MPTP-) control different ions in and out of mitochondria and participate in the process of apoptosis. However, the specific mechanism is not clear. Mitochondrial DNA (mtDNA) is vulnerable to damage due to its structural characteristics, resulting in mutations and deletions of mtDNA, which becomes one of the controlling factors of apoptosis. It is also an important component of organism. Ruthenium Red RR can inhibit the calcium channel in mitochondria. To investigate the effects of no and RR on mtDNA copy number and MPTP opening, it is helpful to explore the specific mechanism of no on postharvest storage of peach fruit in Feicheng peach. It is important to increase the storage time of peach fruit. In this experiment, the primer of NAD1 gene was obtained from National Center for Biotechnology Information (NCBI), which was obtained from National Center for Biotechnology Information Center (NCBI) of Fecheng peach. Using the total DNA of peach fruit under different temperature and different treatments as template, the changes of mtDNA copy number were observed by real-time fluorescence quantitative PCR and fluorescence microscope. The content of ROS, antioxidant enzyme activity, mitochondrial membrane potential and mitochondrial permeability in mitochondria were measured by 15 渭 mol / L ~ (-1) no _ (5 渭 mol) ~ (-1) mol ~ (-1) -PTIO alone and mixed with 20 渭 mol / L ~ (-1) ruthenium red. To study the effect of no on mitochondrial MPTP. The target gene fragment of 159bp was successfully obtained. From the results of real-time fluorescence quantitative PCR and fluorescence microscope of NAD1 gene, we can see that at 0 鈩，

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