干燥条件下赤毛念珠铁蛋白的差异表达及基因克隆

W. Liang, Guangling Jiao, Youwen Zhou, Yaping Zhang, Wei Chen
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摘要

鞭毛藻是一种陆地固氮蓝藻,具有较强的干旱适应性。我们采用了2-DE分析、凝胶图像分析和MALDI-TOF-TOF/MS来比较和鉴定干燥条件下的N.flagelliforme的差异蛋白。结果表明,在干燥胁迫下,铁蛋白、Dps家族蛋白的表达量逐渐下降。利用设计的退化引物,根据鉴定的氨基酸序列克隆铁蛋白基因。获得全长540 bp的DNA (GenBank访问号:HM854287)。同源性分析表明,N.flagelliforme铁蛋白基因具有较高的一致性区域。铁蛋白二级结构由α螺旋和无规螺旋组成。RT-PCR结果显示,干旱胁迫下,铁蛋白mRNA逐渐下降。铁蛋白基因在大肠杆菌中表达,得到22.4 kD的异源蛋白。研究结果为进一步了解极端干旱环境下N.flagelliforme的抗旱分子机制和保护机制奠定了基础。
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Differential Expression and Gene Cloning of Ferritin from Nostoc flagelliformeSubjected to Desiccation
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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