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题名: 集胞藻PCC6803适应寒冷的遗传学基础
作者: 殷春涛
答辩日期: 2004-12-19
导师: 徐旭东
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 集胞藻PCC6803 ; 寒冷光照 ; 低温强化 ; 转录组学 ; 耐寒基因
其他题名: Genetic bases for the adaption of Synechocystis sp. PCC6803 to cold environments
摘要: 蓝藻往往在内陆富营养化水体中形成水华,甚至释放毒素,成为世界性的环境问题,引起公众关注。温带和亚热带地区的蓝藻如何度过冬季寒冷条件对于理解次年水华的发生是十分重要的,而蓝藻生物学中有关0~15℃的研究却十分缺乏。在生物演化史上,蓝藻被认为是植物叶绿体的祖先,被子植物拟南芥基因组中约有18%的基因来自于蓝藻,所以蓝藻与高等植物在生理和遗传的许多方面有相似之处,蓝藻的抗寒基因可能成为农作物遗传改造的基因资源。 集胞藻(Synechocystis sp. )PCC6803是一种单细胞嗜中温蓝藻,在BG11培养基中最适生长温度为30℃,在25~40℃温度区间较适合其生长。本文研究了集胞藻PCC6803在5~15℃低温下的生理特征,发现了低温强化现象;经基因组插入诱变获得对寒冷光照敏感的突变株,鉴定了一些与集胞藻适应低温有关的基因;利用基因芯片技术研究了集胞藻在低温条件下的转录谱,并对在低温培养时上调的基因对于低温强化的作用进行了分析,以期揭示温带和亚热带地区蓝藻越冬的机理。 主要研究内容有如下几个方面: 1、集胞藻PCC6803在低温下的生理特征。在5℃集胞藻PCC6803不能生长,将30℃培养的藻转入5℃在黑暗中可保持存活达2个月以上,而施加100μE.m-2.s-1的光照则存活时间缩短到10天以内。经过15℃培养后再转入5℃处理,无论在100μE.m-2.s-1的光照或黑暗下其耐受力明显提高,作者称这种现象为低温强化。 2、与集胞藻PCC6803适应低温有关基因的鉴定。以卡那霉素抗性标记对集胞藻PCC6803进行了随机或定向插入诱变,在30℃培养并转入5℃、100μE.m-2.s-1的光照下处理,筛选获得了19个寒冷敏感突变株,经过反向PCR和测序确定了被插入的基因。这些基因中未知功能基因4个,分别为sll0268、sll1913、sll1242和slr0688;已知功能基因8个,分别为sll0158(1,4-α-糖原分支酶,glgB)、sll0726(磷酸葡糖变位酶,pgm)、slr0415(钠氢离子交换泵,nhaS5)、slr1908(膜孔蛋白)、slr0089(γ-维生素E甲基转移酶)、sll0262(酰基脂肪脱饱和酶(Δ6),desD)、sll0273(钠氢离子交换泵,nhaS2)。其中sll0158、sll0726均涉及糖原合成,desD、sll0268、sll0273位于基因组同一区域。通过生理特性研究发现突变株sll0158-和sll0726-对寒冷敏感主要由光照引起,突变株slr0415-、slr1908-、slr0089-对寒冷敏感主要由葡萄糖引起。突变株Δsll0268、sll1913-、sll1242-、slr0688-、desD-、sll0273-特异地对寒冷敏感。对这些基因和下游基因分别定向插入失活,证明只有这些基因的突变导致对寒冷光照敏感,而下游基因无影响。sll0268基因例外,其下游基因sll0269突变也导致寒冷敏感性状。在突变株sll0269-、sll1913-、sll1242-、desD -、sll0273-基因组的整合平台中插入同一野生型基因进行互补实验时可恢复其寒冷耐受性。这些突变株在5℃处理时如果遮去光照则并不敏感;当光强降至15μE.m-2.s-1时,突变株sll0273-、sll1913-恢复生存能力。这些突变株在30℃和20℃下生长正常,对光照的适应与野生型相当,但在15℃下表现不同程度的敏感。将突变株desD -、sll0273-在15℃培养后或突变株Δsll0268、sll1913-、sll1242-在20℃培养后转入5℃、100μE.m-2.s-1的光照下处理,其生存力部分甚至完全恢复,提示低温强化可以弥补某些基因缺陷带来的损害。在低磷培养液中生长的desD- 和 sll1242-对寒冷的敏感程度有部分缓解,但在野生型中难以观察到这种低磷培养的效应。亚适应生长条件可能在某种程度上对寒冷耐受有所作用,但低温强化作用机理应远远不止于此。 3、集胞藻PCC6803的转录组学研究。利用基因芯片技术比较集胞藻PCC6803在30℃和15℃生长条件下以及在15℃生长和15℃生长后转入4℃、100μE.m-2.s-1处理2小时后的转录谱差异。在15℃生长的藻细胞中显著上调的基因有:与碳酸盐和CO2转运有关的基因,磷酸葡糖甘油合成酶基因sll1566,与清除脂肪酸过氧化物有关的基因slr1171, psaK类似基因sll0629, sigma H 基因sll0856以及sll1242和sll0330等许多未知功能基因。模板梯度稀释RT-PCR与基因芯片的结果一致。将15℃培养的细胞转入4℃处理时,藻胆蛋白降解酶基因nblAl和类胡萝卜素合成基因crtB、crtD表达增强,与硝酸盐转运或代谢有关的基因nrtA、nrtB、nirA表达降低,一些调控基因sigD、hik34等表达增强,而另一些如sll1291、sll1292表达降低。 4、集胞藻PCC6803低温强化的机理。通过对15/30℃芯片显示显著上调的基因进行定向敲除,分析上调基因在低温强化中的作用,发现sll1566对低温强化基本没有作用,sll0629和sll0330有显著的作用,slr1171 和desB有作用但不稳定。 本研究得到的主要结论有: 1、5℃、100μE.m-2.s-1处理引起30℃培养的集胞藻PCC6803存活率降低,但预先经过15℃低温培养可基本消除其作用,作者称这种现象为低温强化。 2、经插入诱变集胞藻PCC6803和定向敲除获得对寒冷光照敏感的突变株,鉴定了sll1242、desD等与集胞藻适应低温有关的基因。低温强化处理使这些突变株不同程度地恢复对寒冷光照的耐受力。 3、建立了评估单个基因对于低温强化作用的方法,并直接显示了sll0330和sll0629等基因对于蓝藻低温强化的效应。
英文摘要: Cyanobacteria often form waterblooms, even producing toxins, in inland eutrophic waters. It has been a worldwide environmental problem that causes public attention. How cyanobacteria overwinter is very important to understanding of next year’s outbreaks of waterblooms in temperate and subtropical regions. However, little is known about the adaptation of cyanobacteria to low temperatures in the range from 0 to 15℃. In regard to the evolutionary history, a cyanobacterium was proposed to be the ancestor of chloroplasts, and about 18% genes of Arabidopsis nuclear genome were found to be derived from the cyanobacterium. Consequently, there are many similarities between cyanobacteria and higher plants in physiological and genetic characters. Cold tolerance genes of cyanobacteria could be a resource of genes for crop genetic engineering. Synechocystis sp. PCC6803 is a unicelluar mesophillic cyanobacterium, with its optimal growth temperature at 30℃ and a range of favorable temperature from 25 to 40℃. In this research, the author studied the physiological characters at low temperatures from 5~15℃ and found the phenomenon of low temperature enhancement, obtained chill and light-sensitive mutants by random insertion of the genome and identified some genes required to low temperature adaptation. Employing DNA microarray techniques, the author analyzed the changes in transcriptome in response to lowered temperature and the role of genes upregulated at 15℃ in the low temperature enhancement, in the hope to elucidate the mechanism of autumnal acclimation before overwintering of cyanobacteria in temperate and subtropical regions. The experimentation and results are described in 4 parts: 1. The physiological characters of Synechocystis PCC 6803 at low temperatures. There is no growth at 5℃ and cells grown at 30℃ could survive for up to over 2 months in the dark but within 10 days under illumination of 100μE.m-2.s-1. Transfer of cells grown at 15℃ to 5℃, however, would result in a remarkable increase in cold tolerance no matter with or without light, to which I propose to call ‘low temperature enhancement’. 2. Identification of genes required to cold tolerance in Synechocystis sp. PCC6803. After random insertion of the genome or targeted gene disruption of certain genes with a kanamycin-resistance cassette and targeted genes disruption, mutant clones grown at 30℃ were transferred to 5℃ with light of 100μE.m-2.s-1, 19 cold-sensitive mutants were obtained and the inserted genes were determined by inversed PCR and sequencing of the PCR products. Among them, 4 genes sll0268, sll1913, sll1242 and slr0688 encode unknown or hypothetical proteins, 8 genes sll0158(1,4-alpha-glucan branching enzyme, glgB), sll0726(phosphoglucomutase, pgm), slr0415(Na+/H+ antiportor, nhaS5), slr1908(probable porin), slr0089(gamma-tocopherol methyltransferase), sll0262(6 acyl-lipid desaturase, desD) and sll0273(Na+/H+ antiporter, nhaS2) are similar to known genes. Two genes sll0158 and sll0726 are involved in glycogen synthesis, 3 genes desD, sll0268 and sll0273 are located in the same region of the genome. Physiological studies of the mutants indicated that the sensitivity of sll0158- and sll0726- were mainly caused by light and that the sensitivity of slr0415-, slr1908- and slr0089- to cold requires the presence of glucose in the medium, while Δsll0268, sll1913-, sll1242-, slr0688-, desD- and sll0273- were specifically sensitive to cold. Targeted insertion of these genes and their downstream genes showed that the cold-sensitive phenotype were not due to a polar effect, except for sll0268, whose downstream gene sll0269 was also required for cold tolerance. Integration of the wild type genes into a platform in the genome of sll0269-, sll1913-, sll1242-, desD- and sll0273- respectively restored their cold tolerance. In the dark, these mutants were insensitive to cold. When light intensity decreased from 100μE.m-2.s-1 to 15μE.m-2.s-1, mutants sll1913- and sll0273- partially restored their viability. These mutants grow as the wild type at 30℃ and 20℃, but unable to grow or grow poorly at 15℃. Growth of sll1242- and sll0273- at 15℃ or Δsll0268, sll1913-, sll1242- at 20℃ before transfer to 5℃ with light of 100μE.m-2.s-1 could partially or completely restore their survivability, which implies that low temperature enhancement could alleviate the detrimental effects of defects in certain genes. Growth of desD- and sll1242- in a low phosphate medium could partially alleviate their sensitivity to cold, but the effect of such low phosphate growth on the wild type was hardly visible. Suboptimal growth of a cyanobacterium may enhance cold tolerance to some extent, but the mechanism of low temperature enhancement should be much more than the effect of suboptimal growth. 3. Genome-wide transcriptional analyses of Synechocystis sp. PCC6803. Using DNA microarrays, differences of genome-wide transcriptional patterns were analyzed in cells grown at 30℃ and 15℃, or grown at 15℃ but transferred to 4℃ with light of 100μE.m-2.s-1 for 2h. Among genes upregulated in cells grown at 15℃ (compared with 30℃) are: genes involved in transport of carbonate and CO2; sll1566 encoding glucosylglycerolphosphate synthase, slr1171 encoding NADPH-dependent fatty acid peroxidase, sll0629 encoding an analog of PsaK, sll0856 encoding sigma factor H and some genes of unknown functions such as sll1242 and sll0330. RT-PCR with serially diluted templates showed similar results as DNA microarray analyses. Upon transfer of cells grown at 15℃ to 4℃, nblAl encoding a phycobilisome-degrading protease, crtB and crtD involved in carotenoid synthesis were upregulated, some genes required for transport or metabolism of nitrate were downregulated, some regulatory genes such as sigD and hik34 were upregulated but others such as sll1291 and sll1292 were repressed. 4. The mechanism of low temperature enhancement. Through targeted disruption of genes found to be upregulated with 15℃/30℃ microarrays, their roles in low temperature enhancement were analyzed. sll1566, the glucosylglycerolphosphate synthase gene showed no effect on it, while sll0629 and sll0330 showed remarkable effects on it. The role of slr1171 and desB in low temperature enhancement appeared to be wobbling. The main conclusions of this study are as follows: 1. Exposure of cells grown at 30℃ to 5℃ with light of 100μE.m-2.s-1 caused decrease in viability of Synechocystis sp. PCC6803, growth at 15℃ before transfer to 5℃ could basically eliminate the effect of chilling under light. To this phenomenon, the author proposes the name ‘low temperature enhancement’. 2. By insertion mutagenesis of Synechocystis sp. PCC6803, chill and light-sensitive mutants were obtained; genes involved in cold adaptation were identified, including sll1242 and desD. Low temperature enhancement could more or less restore the tolerance of these mutants to chill and light conditions. 3. This study proposed a method to evaluate the role of single gene in the low temperature enhancement and directly showed the effects of sll0330, sll0629 and other genes on it.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/11926
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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集胞藻PCC6803适应寒冷的遗传学基础.殷春涛[d].中国科学院水生生物研究所,2004.20-25
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