中对长小蠹PparOBP1基因的序列分析与原核表达

Sequence Analysis and Prokaryotic Expression of PparOBP1 in Platypus parallelus

为研究中对长小蠹(Platypus parallelus)气味结合蛋白PparOBP1的功能,通过对中对长小蠹转录组数据分析得到中对长小蠹PparOBP1基因cDNA序列,利用生物信息学方法分析PparOBP1基因序列、其编码蛋白的结构、系统进化等相关信息。通过原核表达及蛋白纯化方法,初步探索PparOBP1基因的原核表达情况。结果显示,中对长小蠹PparOBP1开放阅读框(open reading frame,ORF)长399 bp,共编码132个氨基酸,其N基端具有20个氨基酸残基的信号肽;PparOBP1包含4个保守的半胱氨酸位点,属于Minus-C OBP。系统进化分析结果表明,PparOBP1与红棕象甲(Rhynchophorus ferrugineus)的RferOBP2和RferOBPu1亲缘关系较近。蛋白电泳结果显示,PparOBP1蛋白在温度为28℃、诱导时间9 h和IPTG浓度0.1 mmol/L的条件下表达量最高,并且以可溶蛋白形式存在。为进一步利用PparOBP1基因筛选中对长小蠹行为调控剂,明确中对长小蠹气味识别机制奠定理论基础。

In order to study the function of PparOBP1,based on the transcriptome database of Platypus parallelus,the cDNA sequence of the PparOBP1 gene was obtained.With the method of bioinformatics PparOBP1 gene sequence,protein structure,system evolution,and so on were analysed.We preliminarily explored the expression of the PparOBP1 protein by the prokaryotic expression and purification.The results showed that The open reading frame(ORF)of PparOBP1 was 399 bp and encoded 132 amino acids.The signal peptide at the N-terminus was consist of 20 amino acid residues.PparOBP1 contained four conserved cysteine sites,which was consistent with the characteristics of Minus-C OBPs.The results of system evolution analysis showed that PparOBP1 had close genetic relationship with RferOBP2 and RferOBPu1 of Rhynchophorus ferrugineus.The results of SDSPAGE analysis showed that the expression of soluble PparOBP1 protein was highest when the temperature was28℃,the induction time was 9 h and the IPTG concentration was 0.1 mmol/L.It’s helpful to provide a theoretical basis for further study on the molecular mechanisms of chemical communications of P.parallelus.