发状念珠藻醛酮还原酶基因NfAKR的克隆与表达

Gene Cloning and Expression of NfAKR from Nostoc flagelliforme

以发状念珠藻细胞为试材,采用PCR技术克隆了醛酮还原酶基因的开放阅读框(ORF)序列,命名为NfAKR。对基因序列特征进行了生物信息学分析,根据其编码氨基酸序列预测了NfAKR蛋白的三维结构,同时探讨了PEG-6000胁迫下NfAKR的表达特性。结果表明:NfAKR基因的编码序列长912bp,编码304个氨基酸,预测其编码蛋白的相对分子量为33.51kD,理论等电点为4.94,具有醛酮还原酶超家族保守结构域。NfAKR蛋白主要由10个α-螺旋和11β-折叠组成,中间形成一个疏水穴,作为酶的催化活性中心。NfAKR与点形念珠藻处在同一进化枝上,具有较近的亲缘关系。qRT-PCR分析显示,PEG-6000胁迫下NfAKR基因上调表达,当PEG-6000浓度为8%时,其相对表达量为5.66并达到峰值。依据NfAKR基因响应干旱胁迫上调表达的特性,推测醛酮还原酶可能参与发状念珠藻抵御干旱胁迫过程。

Full-length of open reading frame sequence encoding aldo-keto reductases was cloned from Nostoc flagelliforme cells with PCR.The gene was named as NfAKR.Sequence analysis showed that the complete open reading frame of NfAKR was 912 bp,which encoded 304 amino acids residues.The relative molecular mass of NfAKR was 33.51 kD,and its isoelectric point was 4.94.NfAKR protein had the aldo ket red superfamliy domain,and the three-dimension structure was composed by 10α-helices and 11β-sheets,among which there was a hydrophobic cavity as catalytic active site.Phylogenetic analysis showed that aldo-keto reductase from N.flagelliforme and N.punctiforme had high similarity.Quantitative real-time PCR analysis showed that the expression of NfAKRgene was up-regulated under drought stress of PEG-6000.When the concentration of PEG-6000 was 8%,the relative expression was 5.66,reaching the peak value.The NfAKRexpression was upregulated,suggesting that aldo-keto reductase plays a certain role in the process of resisting drought stress in N.flagelliforme.