| 摘要: |
| 【目的】 敲除鱼腥藻PCC 7120的alr0267基因，并对其功能进行初步研究。【方法】 克隆获得鱼腥藻PCC 7120 alr0267基因部分片段，通过构建敲除载体，使其与目的基因发生单交换同源重组，从而将鱼腥藻PCC 7120中的alr0267基因敲除，并对敲除体进行纯化和PCR鉴定，然后对alr0267敲除体和野生型固氮异形胞进行形态观察和统计，同时采用实时荧光定量PCR，在培养不同时间（0，3，8，24 h和10 d）检测野生型和alr0267基因敲除体中与异形胞功能相关基因all0813、all2736、alr2887的相对表达量。【结果】 鱼腥藻PCC 7120 alr0267基因被成功敲除；在缺氮条件下培养6～12 d，敲除体异形胞营养细胞数的均值明显少于野生型；实时定量PCR分析表明，野生型中all0813、all2736、alr2887基因表达量均在培养24 h达到最大，敲除体中这3个基因最大相对表达量的出现时间均有所延迟。【结论】alr0267基因敲除导致异形胞分化频率增加，同时影响异形胞的正常发育。 |
| 关键词: 鱼腥藻PCC 7120 alr0267基因 异形胞 单交换同源重组 实时荧光定量PCR |
| DOI： |
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| 基金项目:广东省自然科学基金项目（S2013010013184）；公益性行业（农业）科研专项（201003021）；国家自然科学基金项目（50977041） |
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| Inactivation and functions of gene alr0267 in cyanobacterium Anabaena sp.PCC 7120 |
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CAO Bipu,FU Xuelin,CAI Xiaodan,et al
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| Abstract: |
| 【Objective】The gene alr0267 was knocked out in Anabaena sp.PCC 7120 and its function was studied preliminarily.【Method】The fragment of alr0267 gene of Anabaena sp.PCC 7120 was cloned and knock-out vector was constructed.The alr0267 gene was inactivated by single crossover homologous integration.The mutant of alr0267 gene was purified and identified by PCR.Then,the morphology of nitrogen-fixing heterocyst from both the mutant and wild-type was observed and counted.The relative expression of related genes all0813,all2736 and alr2887 in wild-type and the mutant were measured at different incubation times (0,3,8,24 h and 10 d) using real-time PCR.【Result】The alr0267 gene of Anabaena sp.PCC 7120 was inactivated successfully.After 6-12 days nitrogen-absent culturing,the average number of vegetative cells in heterocysts of mutant was significantly less than that in wild-type.The maximum relative expressions of all0813,all2736,and alr2887 in the wild-type were detected by real-time PCR 24 h after nitrogen-absent culturing while the time was delayed in the mutant.【Conclusion】The inactivation of alr0267 gene increased the frequency of heterocyst and affected the development of heterocyst. |
| Key words: Anabaena sp.PCC 7120 alr0267 gene heterocyst single-crossover homologus integration real-time PCR |
