【作者】 胡胜；

【导师】 张承才；

【作者基本信息】 华中农业大学 ， 微生物学， 2015， 博士

【摘要】 细胞周期与细胞分化一直是生命科学和医学领域中最基础而重要的研究课题。一个特定细胞的分化通常需要跳出细胞周期,“细胞只能来源于细胞”是对细胞周期与细胞分化的关系的精妙概括和前瞻预测。多年来,细胞周期、细胞分化等基本生命过程的细节及其涉及的大量基因不断被发现和分析,但对这些过程之间的的关系和协调机制却一直没有取得特别重要的突破。多细胞丝状的鱼腥蓝细菌Anabaena sp.PCC7120在环境中缺乏化合态氮元素时,菌丝上一部分营养细胞会脱离细胞周期分化为执行固氮功能的异形胞。这种异形胞的分化是已知的最为古老的细胞分化形式之一,对其机理的探究对理解细胞周期和细胞分化等基本生命过程及其关系有着特殊的意义。本研究以鱼腥蓝细菌Anabaena sp.PCC7120为研究对象,从DNA复制与修复的角度分析细胞周期与异形胞分化的关系。研究中分别系统地记录了细胞周期和异形胞分化过程中的细胞形态变化、DNA与光合色素的分布变化;观察了 DNA复制起始蛋白DnaA、DNA聚合酶Pol I、DNA双链损伤应答蛋白RecN、细胞骨架蛋白MreB、细胞分裂蛋白FtsZ等蛋白质在细胞周期和异形胞分化过程中的亚细胞定位;分析了 DNA复制过程和DNA修复过程之间、细胞周期和异形胞分化之间的协调关系。取得的一些初步结果如下:鱼腥蓝细菌的拟核是细胞内一个相对独立的包被空间。在鱼腥蓝细菌的细胞周期中,营养细胞分裂时细胞质会早于拟核彻底分离;DnaA在hetR突变体中通常定位于细胞中心位置,而在细胞分裂时定位于细胞分裂轴位置;Pol I在细胞中始终与染色体共定位,且在菌丝上表现出表达量的起伏;RecN在营养细胞中表现为一个可以移动的聚集点,在细胞分裂时移动到细胞分裂轴位置;MreB在细胞中以经线状定位于细胞膜下,在细胞分裂时形成直线贯穿于两个子细胞之间;FtsZ位于细胞中部或细胞分裂板位置形成Z环。在异形胞分化过程中,菌丝末端细胞更易分化异形胞;光合色素会降解,染色体会先形成一个“X”或“Y”型结构,在异形胞成熟后因受到极点结构挤压而呈现条带状分布;其极点位置伴随藻青素颗粒常常会出现少量染色体,并贯穿于异形胞和营养细胞之间;Pol I在缺氮6个小时转录水平上升到原来的2倍,其在异形胞中转录水平也高于营养细胞,但是在蛋白质水平上随着时间的延长会下降;RecN在异形胞中同样呈现为一个随机分布的聚集点,但随着缺氮时间的延长迅速降解,在hetR突变体中,缺氮时RecN通常定位于细胞中心位置;MreB在异形胞中和营养细胞类似以经线状定位于细胞膜下;FtsZ在前异形胞中也在细胞中心位置形成Z环,但是在成熟的异形胞中会消失。研究还发现,诱导DNA损伤可以抑制异形胞的发育,而在异形胞分化起始以后再诱导DNA损伤会引起异形胞分化的异常。在营养细胞中,DNA的损伤会导致RecN定位于细胞中心位置或在细胞内增加RecN聚集点的个数;而在异形胞中,DNA的损伤不会引起已消失的RecN聚集点重现。本研究中首次发现:鱼腥蓝细菌的拟核是一个相对独立的细胞结构,其在细胞分裂过程中与细胞质的分裂分离不同步。DNA复制过程和DNA修复过程无论是在细胞周期还是在异形胞分化过程中均存在着相互协助;成熟的异形胞中DNA修复系统不完整。异形胞分化分享了细胞周期中DNA复制、Z环形成等细胞分裂过程的一些早期步骤;异形胞实际上可能是由两个没有分开的,未发育完全的细胞分裂子细胞融合特化而来。更多还原

【Abstract】 Cell cycle and cell differentiation are fundamental and important research topics in field of life science and medicine.Cell differentiation usually needs to escape from the cell cycle;"all cells arise only from pre-existing cells" summarizes the relationship between cell cycle and cell differentiation correctly.From last decades,a large number of genes and some aspects of these basic life processes have been found and analyzed,but the relationship and coordination mechanism between them haven’t been explained in detail.Anabaena sp.strain PCC 7120 is a filamentous cyanobacterium,in response to deprivation of combined nitrogen in environment,vegetative cells differentiate specialized nitrogen-fixing cells called heterocysts.Heterocyst differentiation is one of the most ancient forms of cell differentiation.Exploring for better understanding of its mechanism has a special significance to understand basic life processes and their relationship.This study evaluated relationship between cell cycle and heterocyst differentiation,from perspective of DNA replication and repair,changes in cell morphology,distribution of DNA and photosynthetic pigment;subcellular localization of the DNA replication initiation protein DnaA.,DNA polymerase Pol I,DNA double-stranded damage response protein RecN,cytoskeleton protein MreB and cell division protein FtsZ and coordination mechanism between DNA replication,repair,in Anabaena sp.PCC7120.Results in this study shows that nucleoid of Anabaena was packaged independent.In the cell cycle of Anabaena,cytoplasm segregated before nucleoid.DnaA was usually located in the center of cell in hetR mutant,appeared at cell division plate during vegetative cell division.Pol I,expression was undulated along the filament and always co-located with chromosome in the cell.RecN formed a separate focal point randomLy within cells during the interphase of cell cycle,and moved to central division between daughter cells during vegetative cell division.MreB assembled areshaped line longitudinally located under cell membrane,and transformed to straight line through two daughter cells during cell division.FtsZ was formed Z ring in the cell division plate as in other bacteriums.In the process of heterocyst differentiation,cells at the end of filament were easier to form heterocysts;photosynthetic pigment degraded,chromosome formed an "X" or "Y" type structure first in proheterocyst,then presented zonal distribution in mature heterocyst because of the polar nodule.Normally there is a small amount of chromosomes at polar nodule with cyanophycin granules through cell membrane between vegetative cell and heterocyst.Pol I transcription increases up to two times in 6 hours of nitrogen deficiency,its transcription level in heterocyst was still higher than that of vegetative cell,but decline in extension of time.RecN appeared as a separate focal point in new heterocysts as in vegetative cells,then absent in mature heterocysts,and in hetR mutant cells,at the time of nitrogen deficiency,it was mostly co-localized with nucleoid-occupied regions in central part of cells.MreB location in heterocyst was the same as in vegetative cell.FtsZ formed Z ring in the middle of the proheterocyst;and disappeared in mature heterocyst.In addition,it was found that the DNA damage could inhibit the heterocyst differentiation,and induced DNA damage after heterocyst differentiation could causes abnormal heterocyst differentiation.In vegetative cell,DNA damage leads to an increase of the number of foci and location of RecN in cell center and in heterocyst,while it does not cause reappearance of RecN focal point.According to this research work,in Anabaena sp.PCC7120,it concluded that DNA replication and DNA repair systems are connected and cooperate with each other.Though,DNA restoration system in heterocyst was not complete.Heterocyst differentiation process,first need to start process of DNA replication and Z ring formation in cell division.Based on these results,heterocyst actually develops from immature cell of two daughter cells.更多还原