蜜蜂螺原体细胞骨架相关基因mreB的克隆表达及在螺原体二种形态时的表达差异

【目的】在大肠杆菌中克隆表达蜜蜂螺原体细胞骨架相关基因mreB1?5,并预测所编码蛋白的理化性质,分析这些基因在螺原体螺旋状和非螺旋状时的表达水平,为进一步分析该基因的功能奠定基础。【方法】通过PCR扩增,从Spiroplasma melliferum CH-1基因组中获得mreB1?5基因,构建的重组表达载体pETmreB1?5分别在大肠杆菌BL21中诱导表达,利用镍亲和树脂纯化重组蛋白,通过在线工具预测MreB蛋白质的理化性质和功能域。利用Real-Time PCR比较螺原体CH-1在两种不同形态时mreB1?5基因的表达量。【结果】成功克隆到5个mreB基因,并在大肠杆菌BL21中高效表达。MreB蛋白分子量分别为36、23、23、37和25 kD,可能均为疏水性的蛋白,属于MreB/Mbl蛋白质家族。荧光定量PCR结果显示,螺原体在非螺旋状时mreB1?5基因的表达水平均远低于在螺旋状时基因的表达水平。【结论】本文第一次克隆表达了螺原体细胞骨架相关基因mreB1?5,初步表明这些基因在螺原体形态方面可能具有重要作用,为后续研究螺原体mreB基因在其运动和形态方面的功能提供了重要信息。

Cloning and expression of cytoskeleton-related gene mreB and expression differences between two shapes of Spiroplasma melliferum

Objective] mreB1-5, the cytoskeleton-related genes of Spiroplasma melliferum CH-1 were cloned and expressed in Escherichia coli. Then their proteins properties were predicted. Expression levels of these genes in spiral and non-spiral shape of spiroplasma were determined. Methods] Coding sequences of mreB1-5 were amplified from the genomic DNA of S. melliferum CH-1. Then recombination plasmids pETmreB1-5 were expressed in E. coli BL21 respectively after IPTG induction. Ni-NTA was used to purify the recombinant proteins. The properties of MreB proteins were predicted by online tools. Real-Time quantitative PCR method was applied to compare the expression levels of mreB1-5 genes between two obvious different shapes of S. melliferum CH-1. Results] Five mreB genes from S. melliferum were cloned. The molecular weight of five MreB proteins were 36, 23, 23, 37, 25 kD, respectively. They were possibly hydrophobic proteins with no signal peptide and were identified as part of MreB/Mbl protein family. The results exhibited that the expression levels of genes mreB1-5 in non-spiral shape of spiroplasma were relatively lower than that in spiral shape. Conclusion] This is the first report of clone and expression of spiroplasma cytoskeleton-related genes mreB1-5 which encode actin-like protein MreB. It could be inferred that these genes are closely related with spiroplasma morphology. All results contribute to further investigate the function of actin-like protein MreB in spiral shape and movement of spiroplasma.