发状念珠藻过氧化物还原酶NfPrx基因的克隆与表达分析

Cloning and expression analysis of NfPrx gene from Nostoc flagelliforme

过氧化物还原酶(peroxiredoxin,Prx)属抗氧化蛋白超家族,为了探究Prx在发状念珠藻抗逆过程中所具有的生物学功能,从发状念珠藻细胞中克隆了NfPrx基因的完整开放阅读框(ORF),并对基因序列特征进行了生物信息学分析,根据氨基酸序列预测了该蛋白的三维结构,在此基础上构建了基因的融合表达载体并诱导其表达,同时探讨了PEG-6000胁迫下NfPrx的表达特性。结果表明,NfPrx包含612 bp的ORF,编码203个氨基酸,NfPrx具有PRX_Typ2cys结构域,属于硫氧还蛋白超基因家族,与点形念珠藻、丝状蓝藻7507、微毛藻7126、池生念珠藻CENA21等物种的Prx氨基酸序列一致性高达94%以上。NfPrx与点形念珠藻处在同一进化枝上,具有较近的亲缘关系。利用所构建的原核表达载体,经IPTG诱导和SDS-PAGE电泳,证实诱导表达的NfPrx蛋白与预期结果大小一致。qRT-PCR分析结果显示,NfPrx的表达随干旱胁迫程度的增加先增后降,PEG-6000浓度为8%时,表达量达到峰值,根据表达特性,推测过氧化物还原酶可能参与响应发状念珠藻逆境胁迫过程。

Peroxiredoxin（Prx） belongs to the superfamily that decomposes hydrogen peroxide in living cell. In order to explore the biological functions of peroxiredoxin in process of stress tolerance in Nostoc flagelliforme, we cloned the complete open reading frame of the gene NfPrx, and analysed gene sequence characteristics by bioinformatics. Then, the cloned gene of NfPrx was inserted into vector pET32a. The recombinant plasmids pET32a-NfPrx was expressed in the prokaryotic expression system after its transformation into E. coli BL21. Meanwhile, NfPrx gene expression profile was analysed by qRT-PCR after drought stress of PEG-6000. The results showed that the cloned fragment of NfPrx contains a 612-bp ORF which encodes 203 amino acids. Molecular structure analysis revealed that this gene exhibited the typical structures of PRXTyp2cys domain. Protein sequence identity search showed that NfPrx was similar to Prx gene of Nostoc punctiforme, Calothrix sp. PCC7507, Microchaete sp. PCC7126, Nostoc piscinale CENA21 with similarities of above 94%. Phylogenetic tree reconstructed by neighbor joining method clearly showed that NfPrx protein was classified into a subgroup with N. punctiforme. The recombinant plasmid p ET32a-NfPrx was expressed in a prokaryotic expression system after its transformation into E. coli BL21. The result of SDS-PAGE demonstrated that the expressed protein was consistent with the size of expected protein in the prokaryotic expression system. q RT-PCR result showed that the expression of NfPrx gene rose and then dropped with the increased of PEG-6000 concentration, and reached peak at 8% PEG-6000 concentration. It is hypothesized that peroxiredoxin may involve in the process of response to stress resistance in N. flagelliforme.