本文選題：線粒體　切入點(diǎn)：一氧化氮　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：線粒體除了作為細(xì)胞中有氧呼吸和能量制造場(chǎng)所,還能夠參與細(xì)胞中電子傳遞、細(xì)胞凋亡、細(xì)胞代謝等過程,在機(jī)體正常生理活動(dòng)中發(fā)揮重要作用。一氧化氮(nitric oxide,NO)是一種具有脂溶性的生物活性物質(zhì),在生命信息傳遞中作為第一信使參與到植物體中多種生理反應(yīng)。適量濃度的NO還可以通過調(diào)控線粒體的生理功能來影響植物幼苗的生長(zhǎng)、果實(shí)的成熟和貯藏等過程。線粒體通透性轉(zhuǎn)換孔(mitochondrial permeability transition pore,MPTP)控制不同離子進(jìn)出線粒體,參與到細(xì)胞凋亡過程中,但具體的作用機(jī)理并不明確。線粒體DNA(mtDNA)由于自身結(jié)構(gòu)的特點(diǎn)而易受到損傷,造成mt DNA的突變和缺失,成為細(xì)胞凋亡的控制因素之一。Ca~(2+)作為生物體中第二信使,也是生物體中重要的組成成分。釕紅(RR)能夠抑制線粒體中鈣離子通道。探明NO和RR對(duì)mtDNA拷貝數(shù)的影響和對(duì)MPTP開放的影響,有助于探究NO對(duì)肥城桃桃果實(shí)采后貯藏的具體機(jī)理,對(duì)增加桃果實(shí)貯藏時(shí)間有重要意義。本實(shí)驗(yàn)以肥城桃桃果實(shí)為實(shí)驗(yàn)材料。從美國國家生物技術(shù)信息中心(National Center for Biotechnology Information,NCBI)獲取的NAD1基因設(shè)計(jì)引物,以不同溫度和不同處理下的桃果實(shí)總DNA為模板,通過實(shí)時(shí)熒光定量PCR和熒光顯微鏡來觀察mtDNA拷貝數(shù)的變化情況。選取冷害最嚴(yán)重的5℃下的桃果實(shí),用15μmol L~(-1) NO、5μmol L~(-1)c-PTIO單獨(dú)處理和與20μmol L~(-1)釕紅混合處理,測(cè)量線粒體中ROS含量、抗氧化酶活性、線粒體膜電位、線粒體通透性等指標(biāo),來研究NO對(duì)線粒體MPTP的影響。成功得到長(zhǎng)度為159bp的目的基因片段。從NAD1基因的實(shí)時(shí)熒光定量PCR和熒光顯微鏡的結(jié)果中可以看出在0℃和5℃下,外源15μmol L~(-1) NO能夠促進(jìn)桃果實(shí)線粒體拷貝數(shù)的減小,而在常溫下貯藏桃果實(shí)中,外源15μmol L~(-1) NO抑制了線粒體拷貝數(shù)的減小。釕紅處理使0℃和5℃下處理的桃果實(shí)中mtDNA拷貝數(shù)得到增加。在線粒體膜通透性研究中,外源的15μmol L~(-1) NO處理能夠減少桃果實(shí)線粒體中ROS含量而且使線粒體中抗氧化酶(CAT、SOD、POD)的活性得到提高;NO處理還能夠使線粒體的耗氧量下降,進(jìn)而抑制細(xì)胞的呼吸作用;NO處理一定程度上抑制了線粒體膜電位的降低,維持膜電位在高水平,同時(shí)提高了線粒體膜抗性,抑制線粒體膜透性的增加。15μmol L~(-1) NO-20μmol L~(-1) RR處理在短時(shí)間內(nèi)降低了線粒體中ROS的含量,同時(shí)提高了抗氧化酶CAT的活性、抑制膜電勢(shì)降低和膜透通性的開放。所以15μmol L~(-1) NO-20μmol L~(-1) RR處理可以在短時(shí)間抑制線粒體MPTP的開放。結(jié)果表明外源NO能夠抑制線粒體MPTP的開放,維持線粒體內(nèi)環(huán)境的穩(wěn)定,減緩了桃果實(shí)中細(xì)胞凋亡的過程,使肥城桃桃果實(shí)采后貯藏時(shí)間得到增加。
[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，

本文編號(hào)：1605292