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题名: 鱼腥藻PCC7120中一个参与异形胞分化和图式形成的基因丛
作者: 张维
答辩日期: 2008-01-15
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: 鱼腥藻PCC 7120 ; 异形胞分化 ; 图式 ; alr0099基因丛 ; 调控
其他题名: A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp. PCC 7120
摘要: 鱼腥藻PCC 7120是一种丝状固氮蓝藻,在缺氮诱导条件下,沿着丝体每隔约10个营养细胞分化出一个固氮细胞——异形胞,形成半规律的分布图式。异形胞分化的中心问题是,鱼腥藻如何有效地启动异形胞分化以及控制异形胞分化的图式。宁德刚等(2004)发现,alr0099基因插入突变导致不能缺氮生长并且异形胞间距异常。由于alr0099基因下游还有asr0100、alr0101、asr0102、alr0103和asr0104等基因,它们可能处于同一操纵子,因而不能排除转座子的插入对下游基因造成极性效应。 本研究首先通过基因敲除及突变株互补等遗传学手段,确定了alr0099和alr0101分别对异形胞分化起正负调控作用;然后,通过Northern杂交以及报告基因等方法,检测了它们在缺氮条件下的转录以及它们同已知的异形胞分化相关基因之间的调控关系;最后,还通过基因间的双突变以及细菌双杂交等方法,分析了alr0099和alr0101之间以及同其它异形胞分化相关基因之间在功能上的关联。 主要的研究内容及结果分为以下几部分: 第一、通过同源双交换的方法,构建了alr0099基因丛内各基因的插入突变株,观察了各突变株的异形胞分化,并对各突变株进行了互补分析。alr0099突变株异形胞分化延迟或完全丧失,alr0101突变株可形成成串异形胞。alr0099基因和 alr0101基因对异形胞分化分别起促进和抑制作用。 第二、分别利用Northern blot杂交以及GFP和LUXAB的报告监测等手段,检测了alr0099基因丛在缺氮诱导下的转录表达。结果表明,alr0099基因丛中至少存在两个启动子负责转录:一个位于alr0099的上游,转录产物覆盖alr0099基因,或继续转录覆盖下游asr0100和alr0101等;一个位于asr0100的上游,转录产物覆盖asr0100和alr0101。它们均于缺氮诱导后在原异形胞及异形胞中上调表达。 第三、利用GFP报告基因研究了alr0099和alr0101间以及它们同其它已知功能基因之间的调控关系。结果表明,alr0099基因的表达受其自身的抑制,而alr0101则对它有促进作用;alr0099和alr0101基因的转录依赖于hetR,并不依赖于hetC;alr0099在不同位置的插入突变显示对hetR基因表达的不同影响。 第四、构建了双敲除突变株,用以研究两个基因间的上位关系,同时还利用细菌双杂交方法检测了Alr0101和HetR之间的相互作用。结果表明,alr0099的突变对于patS和hetN突变株没有影响,但可抑制alr0101突变株表型;alr0101的突变可使patA突变株恢复形成居间异形胞。Alr0101与HetR没有直接的相互作用; alr0101、patS和hetN在图式形成中各自发挥独立的功能。 通过本研究可以得到以下结论:在鱼腥藻中, alr0099参与启始异形胞的分化,alr0101参与抑制成串异形胞的形成。这两个基因既可各自转录,又可以同一转录本表达,但是它们的产物对于该启动子的调控作用却是正好相反的。正是这种共同表达和调控的方式使鱼腥藻在缺氮诱导过程中按照适当的速度和频率去完成异形胞分化。
英文摘要: Anabaena sp. strain PCC 7120 is a filamentous nitrogen-fixing cyanobacterium. When deprived of nitrogen, approximately 10% of vegetative cells can differentiate into heterocysts along filaments in a semi-regular pattern. Key questions about heterocyst differentiation are (1) how the differentiation is initiated and (2) what controls the formation of the semi-regular pattern. Ning and Xu (2004) reported, an Anabaena mutant with alr0099 disrupted by transposon was unable to grow on dinitrogen and showed abnormal heterocyst pattern. Because the gene cluster asr0098-alr0099-asr0100-alr0101-asr0102-alr0103-asr0104 may be transcribed as an operon, the phenotype of the mutant could be due to a polar effect of the transposon within alr0099 on downstream genes. In this study, investigations using gene knock-out and complementation showed that alr0099 and alr0101 excert positive and negative effects on heterocyst differentiation respectively, Northern blot hybridization and GFP reporter gene were employed to analyze the transcription of these genes upon nitrogen stepdown and the regulatory relationship with other genes invoved in heterocyst differentiation, double mutation and bacterial two-hybrid system were employed to investigate the functional connections between these genes. The researches and results are in four parts: (1) Insertion mutants of alr0099, asr0100, alr0101, asr0102, alr0103 and asr0104 were generated by double homologous recombination, investigated for heterocyst differentiation and confirmed by complementation. Mutants of alr0099 showed delayed or completely abolished heterocyst differentiation, while a mutant of alr0101 produced multiple contiguous heterocysts. The results indicated that alr0099 and alr0101 were required for positive and negative regulation of heterocyst differentiation respectively. (2) The transcription of the alr0099 gene cluster was investigated by using Northern blot hybridization and GFP or LuxAB reporter gene. At least two promoters are involved in the transcription of the alr0099 gene cluster. The first is located upstream of alr0099, initiating the transcription of alr0099 that may extends to asr0100 and alr0101; the second is located upstream of asr0100, initiating the transcription of asr0100 and alr0101. These transcripts are upregulated in proheterocysts or heterocysts after nitrogen stepdown. (3) The regulatory relationships alr0099, alr0101 and other reported genes were investigated using GFP reporter gene. The expression of alr0099 was self inhibited and promoted by alr0101. The transcription of alr0099 and alr0101 were both dependent on hetR but not on hetC. Insertion at different locations within hetR showed different effects on its expression. (4) Double mutants were constructed for investigations of epistatic interactions between genes, and a bacterial two-hybrid system was employed to investigate the interactions of HetR and Alr0101. Results indicated that a mutation in alr0099 did not influence the phenotypes of hetN or patS mutants, but suppressed Mch phenotype of alr0101, and that a null mutation in alr0101 resumed the formation of intercalary heterocysts ina patA mutant. Alr0101 and HetR had no direct physical interaction, and alr0101, patS and hetN played independent role in pattern formation. The main conclusions of this study are as follows: In alr0099 is required for initiating heterocyst differentiation and alr0101 for inhibiting the formation of multiple contiguous heterocysts. These two genes are subject to both independent transcription and co-transcription, and the promoter of alr0099 is under opposite regulation by the two genes. The organization and feedback regulation of the two genes should be part of the mechanism that ensures the formation of heterocysts in proper rate and pattern along filaments.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12016
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
鱼腥藻PCC7120中一个参与异形胞分化和图式形成的基因丛.张维[d].中国科学院水生生物研究所,2008.20-25
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