摘要
本论文主要分三部分:一是利用激光扫描共聚焦显微镜在蓝藻细胞内建立两种活体内荧光成像技术平台:荧光漂白恢复技术(fluorescence recovery afterphotobleaching, FRAP)和荧光共振能量转移技术(fluorescence resonance energytransfer, FRET);二是利用FRAP技术研究与蓝藻状态转换有关的藻胆体的动态扩散及其驱动力和调控因素;三是利用活体内FRET技术初步探讨了蓝藻NAD(P)H脱氢酶复合物的亚基之间的距离及相互作用。
后基因组时代的到来要求对生物大分子尤其是蛋白质的结构、特点及其相互作用的研究由体外转向体内。活体内荧光成像技术的出现使得研究活细胞生理状态下蛋白质的动态变化成为可能。本论文分别利用两种模式蓝藻:Synechococcussp. PCC7942和Synechocystis. PCC6803建立了在蓝藻活细胞内进行FRAP和FRET实验的技术平台,用以研究活细胞生理状态下蓝藻光合膜蛋白的动态变化。
在蓝藻活体内利用FRAP技术的研究已经表明藻胆体可以在类囊体膜上快速的自由扩散,且该动态扩散对蓝藻的状态转换是必需的。但是藻胆体扩散的驱动力及调控因素仍然未知。FRAP是在细胞生物学中被广泛用于测定生物体系动态性包括生物膜组分动态扩散的一种技术,其原理是利用高强度激光将细胞内一小部分区域的荧光分子漂白,然后通过记录该区域随后的荧光恢复,检测周围未漂白区域内的荧光分子与漂白区域荧光分子的交互扩散。本文利用FRAP技术和77K低温荧光发射谱研究了PQ的结构类似物-对苯醌(BQ)对蓝藻Synechococcus sp.PCC7942的状态转换和藻胆体动态扩散的影响。结果表明:BQ在暗中可以诱导蓝藻向状态1转换,且将蓝藻固定在状态1。同时,BQ处理显著影响了藻胆体在类囊体膜上的动态扩散,使多数藻胆体被固定在类囊体膜上,不能进行自由扩散。据此我们推测PQ库的氧化还原状态参与调控藻胆体的动态扩散,并提出了PQ库氧化还原状态调控藻胆体动态扩散及状态转换的假想模型。
NAD(P)H脱氢酶复合物(NDH)是近年来在蓝藻和高等植物等的类囊体膜上发现的另一类膜蛋白复合体,在蓝藻中参与呼吸和循环电子传递及参与CO2的吸收,对于蓝藻许多重要生理过程是必需的。但蓝藻NDH复合物的体外完整分离尚未实现,其完整的亚基组成也存有争议。本论文利用绿色荧光蛋白(greenfluorescent protein, GFP)的变种黄色荧光蛋白(yellow fluorescent protein, YFP)
This work consists of three parts. Firstly, two types of live cell fluorescentimaging techniques -fluorescence recovery after photobleaching (FRAP) andfluorescence resonance energy transfer (FRET) were developed in livingcyanobacteria cells using laser scanning confocal microscope. Secondly, FRAP wasused to detect the diffusion of phycobilisomes on thylakoid membranes and thedriving force (or the regulatory factors) of this diffusion. Thirdly, FRET was used todetect the interaction of two subunits of NAD(P)H dehydrogenase complex incyanobacteria.
The arriving of the post-genome era requires the studying of the structures,characteristics and interactions of biological macromolecules especially proteinsturning from in vitro to in vivo. This study used two model cyanobacteria-Synechococcus sp. PCC7942 and Synechocystis. PCC6803 to develop FRAP andFRET techniques in living cyanobacteria cells, which can be used to study thedynamics of photosynthetic membrane proteins.
Studies using FRAP in living cyanobacteria cells have shown thatphycobilisomes diffuse rapidly on thylakoid membranes, and this diffusion is requiredfor state transitions in cyanobacteria. However, the driving force and the regulatoryfactors of phycobilisome diffusion are yet unknown. FRAP is a technique widely usedin cell biology to observe the dynamics of biological systems, including the diffusionof membrane components. This technique involves the use of a highly-focusedconfocal laser spot to selectively bleach the fluorophores in a small region of the cell.The diffusion of the fluorophores can then be monitored by observing the spread andrecovery of the bleach. In this study, we use FRAP and 77K fluorescence spectra toinvestigate the effects of 1, 4-benzoquinone (BQ), one of the benzoquinone analogues,on state transitions and phycobilisomes mobility in Synechococcus sp. PCC7942. Theresults show that BQ can induce a transition from state 2 to state 1 in the absence ofactinic light and cells treated with BQ were locked in state 1. At the same time, BQaffects the diffusion of phycobilisomes drastically. Most phycobilisomes are
immobilized on thyalkoid membranes. It is suggested that the redox state of PQ poolcan regulate the phycobilisome diffusion in cyanobacteria.NAD(P)H dehydrogenase complexes(NDH) is another photosynthetic membranecomplex found in thylakoid membranes in cyanobacteria and green plants in recentyears. It has specific functions in respiration and cyclic electron flow as well as inactive CO2 uptake. Attempts to isolate intact NDH complexes from cyanobacteria orplant chloroplasts have met severe difficulties due to the fragility of the enzyme andthe low quantity present in the thylakoid membrane. Also, the intact subunitscomposition is yet unknown. In this work, two variants of green fluorescentprotein(GFP)-yellow fluorescent protein(YFP) and cyan fluorescent protein(CFP)were used to label D4 and CupB subunits of NDH complexes respectively. ThenFRET was used to detect the interactions of these two subunits and theconformational changes of NDH complexes in different physiological conditions.Three different methods including sensitized emission, acceptor photobleaching anddonor photobleaching dynamics were used to detect FRET signals. However, theresults showed that many factors can interfere FRET studies in cyanobacteria. Theautofluorescence of the cell is high and the expression level of the fluorescent proteinsis low relative to the cell autofluorecence, which makes the ratio of signal to noise toolow to be detected. In this study, we also found a photo-activation-like phenomenon inthe range of 465-565 nm using 405 nm laser for XYT scanning. This may be relatedto the activity of carotenoids, which can protect the photosystems in the high lightmenace.
引文
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