溶藻弧菌hutB基因的克隆及其在不同铁源下的表达分析

根据已发表的溶藻弧菌(Vibrio alginolyticus)血红素结合蛋白(Periplasmic Hemin-Binding Protein,HutB)基因序列设计引物,PCR扩增溶藻弧菌HY9901株的hutB基因,序列分析结果显示该基因全长870 bp,共编码289个氨基酸,分子量约为30.59 ku,PI为6.45。细胞定位、SignalP4.0、TMHMM Server 2.0和SoftB erry-Psite预测结果显示,hutB位于外周质中,存在信号肽切割位点,没有跨膜结构域,氨基酸序列含有1个cA MP和cG MP的蛋白激酶磷酸化位点,4个蛋白激酶C磷酸化位点等多个活性位点。系统进化树结果显示,溶藻弧菌hutB与坎氏弧菌(Vibrio campbellii)和哈氏弧菌(Vibrio harveyi)聚为一簇。qR T-PCR技术初步探究hutB在不同铁源下的表达量,结果表明,溶藻弧菌hutB在含有FeC l3的富铁培养基中,表达量与对照组相比差异不显著;在含有血红素的条件下表达量上调;在同时含有2-2'二联吡啶和血红素时上调极显著(P0.01);在含2-2'二联吡啶的铁限制环境下,表达量下调极显著(P0.01)。

Molecular cloning of periplasmic hemin-binding protein (HutB) gene from Vibrio alginolyticus and its expression pattern under various iron sources

Primers for PCR cloning were designed according to periplasmic hemin- binding protein(HutB)gene sequence of Vibrio alginolyticus published in GenBank. The hutB gene of V.alginolyticus strain HY9901 was amplified by PCR and cloned into pMD18-T vector. Sequence analysis revealed that hutB gene is 870 bp and encodes a putative protein of 289 amino acids. The predicted molecular weight (MW) of HutB was 30.59 kD with an estimated pI of 6.45. Using SignalP 4.0 and TMHMM Server 2.0 software, it was predicted that the HutB protein was located in periplasmic. It contained a signal peptide cutting site, but did not have transmembranous region. This protein had one cAMP- and cGMP- dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, and so on. To further analyze the evolutionary relationship among HutB, a molecular phylogenetic tree was constructed using Mega 5.0 software. In this tree, the HutB protein showed high genetic relationship with Vibrio campbellii and Vibrio harveyi. Using qRT-PCR, the expression changes of hutB under various iron sources were examed. Cultured in iron-rich medium containing FeCl₃, the expression levels of hutB changed a little compared with the control group. However, in the medium with hemin, the expression level of hutB increased, specifically in the iron-restricted environment exists with hemin, the expression of hutB increased significantly(P<0.01). At the same time, the expression of hutB under iron limiting environment with: 2,2'-Dipyridyl was lower than control group(P<0.01).