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题名: 裸藻质体在竞争和适应中的作用
作者: 王江新
答辩日期: 2002
导师: 徐旭东 ; 施之新
专业: 遗传学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
其他题名: The role of euglenoid plastids in competition and adaptation
摘要: 裸藻类中并存着光合自养、腐生和吞噬三种不同营养类型。具有色素体的裸藻既能够在光下进行光合作用,也能在黑暗条件下利用有机质生长。无光照时裸藻色素体不发育,以原质体的形式存在。无色、腐生型的长变胞藻具有原质体结构和质体DNA的残余,永久性地失去了由原质体发育成为色素体的能力。长变胞藻原质体结构的生理功能尚属未知。RAPD和1 85 rRNA序列分析结果都显示长变胞藻和纤细裸藻有着密切的亲缘关系。本研究利用ofl0Xacin处理得到了性状稳定的纤细裸藻退色突变株,光学显微镜观察、色素吸收光谱扫描及叶绿素自发荧光、DAPI荧光染色、全细胞蛋白质SDS-PAGE和电子显微镜观察证明细胞中无质体或类囊体结构,不存在DAPI荧光染色可以检测到的质体DNA,无叶绿素的合成,全细胞蛋白质组成也发生了改变。为比较完全失去质体结构的突变株和野生型的适应力,我们对二者在不同条件下的生长特性进行了研究,结果发现野生型与突变株的生长在有机质丰富的培养液中差别微弱,质体的丢失并不产生显著影响。根据以上研究结果,设置了光照与黑暗环境以及不同的培养基浓度条件下突变株与野生型之间的生长竞争实验。在光照情况下,当营养物质成为限制因子时,由于具有光合能力野生型生长受影响较小,在与没有光合器的突变株的竞争中很快地显示出优势;在黑暗中二者均只能以异养方式生长,但突变株仍然处于竞争弱势,显示原质体结构对于生存竞争的作用。在先期实验中以SDS-PAGE观察到野生型和突变株各有特异表达的蛋白带。为探明质体在光合作用以外的其它生理功能,检查了核编码基因的转录表达差异。发展了一种快捷的差异显示技术-选择性cDNA3'-端片段的差异显示(Selective Difterential display of3'-end restriction fragments of cDNA),在我们的条件下能够以普通EB染色的琼脂糖凝胶电泳直接观察到一些可能反映差异表达的PCR产物带。尝试了10条(T15)cNN选择性引物共观察到15条差异显示的DNA带。将这些带切割、纯化、再扩增,并克隆、测序。根据其序列设计引物以RT-PCR检查这些墓因转录表达水平的差异。结果证实其中至少有3个在无质体的突变株中转录水平有显著提高,将野生种和突变株在光照或黑暗培养时均如此。原质体在竞争中可能以两种方式发挥作用:1)在原质体内进行的某种反应对异养代谢或细胞分裂有微弱促进作用;2)原质体调控某些核基因并影响细胞的生理状态,从而间接地对异养代谢效率或增殖产生微弱影响。SDS-PAGE与选择性cDNA3'立湍片段的差异显示结果都表明,失去质体之后,细胞的蛋白质组成和细胞核的基因表达发生了改变。这说明细胞核和质体之间的作用是相互的:质体的发育受细胞核的调控,另一方面,某些核基因的表达也受到质体的控制。在质体被去除时,细胞核中某些基因表达增强,以适应代谢类型的变化。或许正由于原质体结构在竞争适应中的效应,长变胞藻和自然界某些裸藻褪色种仍保留有这一结构。本文主要有两点突出之处:1)应用裸藻褪色突变株与野生型的生长竞争实验探讨了原质体对于裸藻的适应意义,发现在黑暗环境和异养生长条件下原质体对于细胞的生存竞争有重要作用;2)改进了一种差异显示技术并利用该技术来研究质体的有无对基因表达的影响,证明质体丢失可导致某些核基因表达的显著变化。
英文摘要: Among euglenoid species, three nutrition types coexist, the phototrophic, saprophytic and phagotrophic. Euglenoids possessing chloroplasts are capable of photosynthesis in the light and growing on organic substances in the dark. Without light, euglenoid chloroplasts are not developed, but remain in the form of proplastid. Astasia longa, a colorless and saprophytic species that retains its proplastid structure and a relic of plastid DNA, permanently loses the capability to develop chloroplasts from proplasts. The physiological function of proplastids in A. longa is kept unknown. Analyses by RAPD and 18S rRNA sequence both support that A. longa is closely related to Euglena gracilis.In this research, a stable plastid-less mutant of E. gracilis was generated by treatment with ofloxacin. Light microscopy, absorption spectra of pigment extracts, auto-fluorescence of chlorophyll, DAPI-staining fluorescence microscopy, SDS-PAGE of whole cell proteins and transmission electron microscopy showed that the mutant cells have neither plastid nor thylakoid membrane structure, nor DAPI-detectable plastid DNA, and chlorophyll synthesis, and that whole cell protein composition was changed.To make comparison of adaptability of the plastid-less mutant and the wild type strain, we studied their growth characters under different culture conditions, found that the difference of their growth in a medium rich in organic substances are hardly detectable and that the loss of plastid exerts only a weak effect, if any, on the heterotrophic growth. Based on these results, we designed different conditions combining light or dark with medium concentration for tests of competition between the wild type and mutant strains. When grown in the light and with nutrition as thein limiting factor, the wild type was less influenced due to its capability of photosynthesis and quickly became predominant over the mutant that has no photosynthetic apparatus. However, when grown heterotrophically in the dark, the mutant remained less competitive than the wild type, showing that the euglenoid proplastids play a role in competition.In the previous experiments of SDS-PAGE, we observed that the wild type and mutant strains each had specifically expressed protein bands. To explore the physiological functions of plastids other than photosynthesis, we examined the differences in transcription of nuclear genes. A quick procedure for differential display technology was developed, namely, the selective differential display of 3'-end restriction fragments of cDNA. Under our conditions, PCR products that may reflect the differential expression could be observed by regular agarose electrophoresis and EB-staining. With 10 selective primers (T^CNN, totally 15 DMA bands resulting from the differential display were obtained. These bands were excised from the gel, purified and re-amplified, then cloned and sequenced. Based on the sequences, primers were designed to inspect the differential transcription of these candidate genes by RT-PCR. The results showed that at least 3 of these genes had remarkably increased transcription in the plastid-less mutant, no matter the wild type and mutant strains were grown under illumination or not.The proplastid may play its role in competition in two different ways. Firstly, some reactions in proplastids promote heterotrophic metabolism or cell division slightly. Secondly, proplastids may control some nucleus-encoded genes and influence the physiological status of the cell, therefore slightly affect the efficiency of heterotrophic metabolism or propagation via indirect routes. The results of SDS-PAGE and differential displays both showed that the composition of cell proteins and the expression of nuclear genes were changed after the loss of plastid. This phenomenon suggests that the interactions between the nucleus and plastids are reciprocal: the development of plastid is controlled by the nuclei, on the other hand, certain nuclear genes by plastids. After removal of the plastid, the expression of some nuclear genes are enhanced to adjust to the change of metabolic types. Perhaps it is due to its role in adaptation that A. longa and other bleached species of euglenoids retained their proplastid structure.Two points of this dissertation deserve being highlighted: 1) competition experiments between the wild type and bleached mutant strains of E. gracilis were applied to investigate the effect of proplastid on adaptability, in which such structure was found to be important to the competitiveness during heterotrophic growth in the dark; 2) a new procedure for differential display technology was developed and used to study the influence of plastid on nuclear gene expression, with which it was shown that the loss of plastid could cause remarkable changes in expression of certain nuclear genes.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12644
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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裸藻质体在竞争和适应中的作用.王江新[d].中国科学院水生生物研究所,2002.20-25
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