嗜水气单胞菌转录调控蛋白基因的克隆与序列分析

Cloning and Sequence Analysis of Transcriptional Activator Protein Gene from Aeromonas hydrophila

根据嗜水气单胞菌(Aeromonas hydrophila)转录调控蛋白(AhyR)基因序列设计特异性引物,采用PCR方法克隆嗜水气单胞菌Zf1菌株AhyR基因,测序目的基因大小为1 063 bp。对AhyR基因编码蛋白进行生物信息学分析,推导其开放阅读框(ORF)编码260 aa,含有自体诱导物结合域(18-168 aa)、LuxR型螺旋-转角-螺旋(HTH)DNA结合结构域(176-241 aa),以及13个磷酸化位点(15S、30T、62S、144S、145S、156S、161Y、173S、184T、188T、200S、227S、231Y)。AhyR蛋白三级结构与模板(PDB:3SZT_A)相似性达28.40%,结果显示LuxR型螺旋-转角-螺旋结构域主要以α螺旋分布在C端外侧,这与拉马钱德兰图(Ramach andran plot)检测分析AhyR蛋白空间结构基本一致。

Transcriptional activator protein（AhyR）gene of Aeromonas hydrophila strain Zf1 was amplified by PCR using the designed primers, resulting in a 1 063 bp DNA sequence. The sequencing analyses revealed AhyR gene encoding a protein of 260 amino acids which has autoinducer binding domain（18-168 aa）and LuxR-type helix-turn-helix（HTH）domain for DNA binding（176-241 aa）. Moreover, AhyR protein was predicted containing 13 phosphorylation sites（ie., 15S, 30T, 62S, 144S, 145S, 156S, 161Y, 173S, 184T, 188T, 200S, 227S and 231Y）. The homology modeling of 3D structure of AhyR protein was carried out based on the template retrieved from chain A of quorum sensing control repressor（PDB：3SZT_A）. AhyR protein showed the homology of 28.40%with the standard model, and the physiological activity subunit LuxR-type HTH domain profile was located in the outer surface of the C-terminal. The quality of the resulting protein structure was further checked by the Ramachandran plot.