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Alternative TitleStudies on the gas vesicle gene cluster in Microcystis sp.
Thesis Advisor徐旭东
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword微囊藻 伪空胞基因丛 结构类型 演化关系
Abstract微囊藻(Microcystis sp.)是广泛分布于世界各地的单细胞蓝藻,多生长于湖泊和池塘中,在夏季易大量生长而形成水华,造成水环境恶化。在微囊藻细胞中,有一种特殊的由中空圆柱状气囊组成的结构——伪空胞能够为细胞提供浮力,使它们在水体中垂直迁移,获得适宜的生长条件,因而,对于微囊藻水华的暴发起到重要的作用。本研究比较了多个微囊藻株的伪空胞基因丛结构特点,探讨了它们的系统演化关系和表达差异,并对一种微囊藻中伪空胞基因丛的转录调控进行了初步研究。 主要研究内容和研究结果分为四个部分: 第一,微囊藻FACHB854伪空胞基因丛的获得。首先构建FACHB854的基因组文库,筛选得到了含有gvpA-gvpC基因的克隆;利用PCR、反向PCR和测序,最终获得包含10个不同的基因、约7.5 kb的基因丛全长序列,其中gvpA有两个拷贝;用Southern blot杂交的方法证明,在FACHB854的基因组中仅存在这一个伪空胞基因丛。 第二,微囊藻伪空胞基因丛的结构类型、演化关系和表达差异。对多种微囊藻的gvpA-gvpC区域进行了克隆测序,总结出伪空胞基因丛的四种结构类型:2gvpA+gvpC(3)、2gvpA+gvpC(4)、3gvpA+gvpC(4)和4gvpA+gvpC(3);以微囊藻的rbcLX区域序列构建系统进化树,同时用各微囊藻株的gvpA-gvpC和gvpA-gvpA间隔区域序列、gvpC基因序列构建进化树分析伪空胞基因丛的进化过程,分析发现伪空胞基因丛的演化过程与微囊藻的系统进化基本同步;电镜观察不同微囊藻气囊的形态,进一步证明GvpC蛋白内部重复单位越多,微囊藻气囊的直径越大,而gvpA基因的拷贝数与气囊的直径无关。 第三,gvp基因转录调控的初步研究。以微囊藻FACHB930为材料,在实验室条件下考察了不同温度、光照强度、氮源、碳源以及pH值条件下藻细胞的漂浮情况,认为该藻株的漂浮情况与培养物的初始pH值有关;用模板梯度稀释RT-PCR证实,在不同初始pH条件下,三个gvpA拷贝和gvpC基因的转录水平存在差异;用实时荧光定量PCR的方法,进一步证实微囊藻FACHB930中三个gvpA拷贝与gvpC基因的转录调节是相互协调的。 第四,在研究中发现了一个可转座的微型插入因子。在微囊藻FACHB854的伪空胞基因丛中发现一个具有正向靶点重复和末端反向重复但不含有转座酶的微型插入因子ISM854-1;Southern blot杂交发现这一微型插入因子及其同源物在微囊藻FACHB 854的基因组中至少存在10个拷贝,而在其他的微囊藻株中没有或极少;用反向PCR的方法获得ISM854-1更多拷贝的序列,并确定其靶位点为8-bp的AT丰富区; ISM854-1的变异体ISM854-1A可能形成复合转座元件进行转座。 本研究得到以下主要结论: (1) 微囊藻的伪空胞基因丛可分为四种基本结构类型:2gvpA+gvpC(3)、2gvpA+gvpC(4)、3gvpA+gvpC(4)和4gvpA+gvpC(3)。其中,gvpA前的数字表示其拷贝数,gvpC后面括号里的数字表示GvpC蛋白中由33个氨基酸残基组成的保守重复单位的数量。 (2) 微囊藻FACHB930的三个gvpA拷贝和gvpC基因的转录水平是相互协调的。 (3) 蓝藻中存在非自主转座的微型插入因子。
Other AbstractMicrocystis is a group of unicellular cyanobacteria distributed all over the world and most frequently occur in freshwater lakes and ponds. In summer, massive production of Microcystis results in the outbreak of waterblooms, causing deterioration of water environments. In Microcystis cells, a special hollow cylindrical structure, gas vesicle, facilitates buoyancy and enables cells to perform vertical migration in water column and gain favorable growth conditions, consequently, plays an important role in the outbreak of Microcystis waterblooms. In this study, gas vesicle gene clusters from various Microcystis strains were compared, their phylogenetic relationship and differences in gene expression were investigated. A preliminary study on the regulation of gvp gene transcription in a strain of Microcystis was also conducted. The main researches and results are in four parts: (1) The sequence of the gvp (gas vesicle protein) gene cluster from Microcystis sp. FACHB854. The genomic library of FACHB854 was constructed and a clone carrying gvpA-gvpC gene was identified. Based on the sequence of this region and the published sequence of Microcystis PCC 7806 while this study was going on, the whole sequence of the 7.5-kb gene cluster was finally obtained using regular PCR, inverse PCR and sequencing. The gene cluster contains 11 genes, gvpA having two copies. Southern blot hybridization showed that only one gvp cluster is present in the genome of FACHB854. (2) The structural types, phylogenetic relationships and coordinate regulation of gvpA-gvpC regions in Microcystis. By comparing the sequences of the gvpA-gvpC regions from 8 Microcystis strains, gvp clusters were classified into four structural types: 2gvpA+gvpC (3), 2gvpA+gvpC (4), 3gvpA+gvpC (4) and 4gvpA+gvpC (3). The phylogenetic trees based on partial rbcLX, intergenic sequences between gvpA copies or gvpA and gvpC, gvpC gene showed that the evolution of gvp cluster was basically consistent with the phylogeny of Microcystis. Measurements by electron microscopy confirmed that the diameter of gas vesicles in Microcystis was correlated with the length of GvpC protein, but not to the copy number of gvpA. (3) A preliminary study on the transcriptional regulation of gvpA and gvpC genes. The effects of various environmental factors, including temperature, light intensity, nitrate concentration and pH value, on the buoyancy of FACHB930 were investigated in BG11 medium, and the results showed that the buoyancy of this Microcystis strain was apparently regulated by the initial pH value. Half-quantitative and quantitative RT-PCR both showed that transcriptional levels of 3 gvpA and gvpC genes changed coordinately with the initial pH value of the culture. (4) A miniature transposable insertion element in Microcystis sp. FACHB 854. A 186-bp miniature insertion element, ISM854-1, was found in FACHB 854, with imperfect terminal inverted repeats, target direct repeats but without any transposase-encoding capacity. Southern blot hybridization showed that ISM854-1 and homologues were present with more than 10 copies in the genome of FACHB 854, but none or few in other Microcystis strains. More ISM854-1 copies were obtained employing inverse PCR, all located at 8-bp long and AT-rich target sequences. A variant of ISM854-1, denoted ISM854-1A, may transpose in pairs in a structure like a composite transposon. The main conclusions of this study are: (1) Gas vesicle gene clusters were classified into four structural types: 2gvpA+gvpC(3), 2gvpA+gvpC(4), 3gvpA+gvpC(4) and 4gvpA+gvpC(3), in which numerals before gvpA are copy numbers of tandem gvpA genes and those in the parentheses are numbers of conserved repeating units (33RR) in GvpC proteins. (2) The transcriptional levels of 3 gvpA copies and gvpC gene were regulated coordinately in the strain FACHB930. (3) A non-autonomous transposible miniature insertion element was found in a cyanobacterium.
Document Type学位论文
Recommended Citation
GB/T 7714
许敏. 微囊藻伪空胞基因丛的研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2006.
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