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作 者 ：梁文裕;焦广飞;周有文;张亚萍;陈伟

期 刊 ：植物科学学报 2012年 30卷 1期 页码：72-78

关键词：发菜；Ferritin；差异表达；基因克隆；RT-PCR；原核表达；

Keywords:Nostoc flagelliforme, Ferritin, Differential expression, Gene cloning, RT-PCR, Prokaryotic expression,

摘 要 ：发菜(Nostoc flagelliforme)是一种陆生固氮蓝藻,具有强烈的旱生生态适应性。运用双向电泳技术、凝胶图像分析、MALDI-TOF-TOF/MS质谱鉴定和数据库检索,发现发菜Ferritin在干旱胁迫条件下表达量逐渐降低。根据鉴定的Ferritin已知氨基酸序列设计简并性引物克隆该基因,获得了长度为540 bp的DNA,GenBank登陆号为HM854287。序列比较显示该基因具有较高的保守性,蛋白质二级结构主要由α螺旋和随机卷曲构成。RT-PCR分析表明,Ferritin mRNA在干旱胁迫条件下表达量逐渐降低,与Ferritin的表达趋势一致。将Ferritin基因在大肠杆菌中表达,获得符合预期的外源重组蛋白(22.4 kD)。实验结果可为进一步研究发菜耐旱的分子机理及探讨发菜对极端干旱环境的适应和保护机制奠定基础。

Abstract:Nostoc flagelliforme is a terrestrial nitrogen-fixing cyanobacterium with strong drought adaptability.We performed 2-DE analysis,gel image analysis and MALDI-TOF-TOF/MS to compare and identify differential proteins from N.flagelliforme subjected to desiccation.The results indicated that Ferritin,Dps family protein expression quantum gradually declined under desiccation stress.The Ferritin gene was cloned by designed degeneracy primer based on identified amino acid sequences.A full length of 540 bp DNA was obtained (GenBank access number: HM854287).Homology analysis showed that the N.flagelliforme Ferritin gene had high consensus regions.The secondary structures of Ferritin were made up of α helix and random coil.The RT-PCR showed that mRNA of Ferritin gradually declined under drought stress.The Ferritin gene was expressed in Escherichia coli,and a 22.4 kD heterologous protein was observed.The results laid a foundation for understanding the drought-resistant molecular mechanisms and protection mechanisms of N.flagelliforme in extreme arid environments.

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