松墨天牛OBP基因MaltOBP2和MaltOBP6的克隆、序列分析及组织表达谱和原核表达研究

【目的】本研究旨在探索松墨天牛Monochamus alternatus Hope在嗅觉识别寄主植物过程中扮演重要角色的气味结合蛋白(odorant binding proteins,OBPs)的结构及功能。【方法】利用生物信息学方法对得到的Malt OBP2和Malt OBP6基因序列和蛋白结构进行分析,并通过实时荧光定量PCR分析Malt OBP2和Malt OBP6在松墨天牛雄虫不同组织和时空中的表达差异,利用p ET32a(+)原核表达载体对Malt OBP2和Malt OBP6进行了诱导蛋白表达。【结果】本研究得到两个松墨天牛气味结合蛋白基因——Malt OBP2(Gen Bank登录号:KP120891)和Malt OBP6(Gen Bank登录号:KP120892),ORF长度分别为402 bp和408 bp,翻译的氨基酸序列均含有4个保守的半胱氨酸位点,表明得到的两个OBP基因的编码蛋白均属于Minus-C OBP亚家族;推导的两个OBP蛋白均有6个α螺旋区域,且α螺旋区域在两个蛋白的位置非常相似,但是两个OBP蛋白推测的配体结合位点和位点极性却完全不同。组织表达模式表明,Malt OBP2和Malt OBP6在成虫头部、触角、下颚(唇)须、腹部末端和足中均有表达,表达程度不一,但都在头部显著表达,触角中的表达量相比其他组织中较低或只是持平。发育表达结果表明,Malt OBP2在蛹触角中的表达量最高,而Malt OBP6在幼虫头部的表达量最高。本研究成功构建了原核表达载体p ET32aMalt OBP2和p ET32a-Malt OBP6,并进行了OBP蛋白诱导表达,低温(16℃和20℃)条件利于蛋白表达在上清液中,延长诱导表达时间(12 h)可以增加蛋白的表达量。【结论】本研究从松墨天牛体内得到了Minus-C OBP蛋白亚家族的两个基因Malt OBP2和Malt OBP6,通过配体结合位点推测它们具有不同的生理功能;通过组织表达谱结果推测这两个OBP基因在松墨天牛中的功能不仅仅局限于嗅觉识别,或还有味觉感受、化学感受等其他生理功能。本研究结果为两个OBP蛋白的结构和功能研究奠定了基础,为探索松墨天牛的化学感受机制提供了条件。

Cloning,sequence analysis,tissue expression profiling and prokaryotic expression of odorant binding protein genes MaltOBP2 and MaltOBP6 from Monochamus alternatus (Coleoptera: Cerambycidae)

【Aim】 Odorant binding proteins (OBPs) play an important role in olfactory recognition process. This study aims to explore the structure and function of odorant binding proteins of Monochamus alternatus. 【Methods】 The cDNA and deduced amino acid sequences of MaltOBP2 and MaltOBP6 genes were analyzed by bioinformatics methods. The expression levels of the two genes in different tissues and developmental stages of M. alternatus were detected by real-time RT PCR. Prokaryotic expression vector was constructed to express the recombinant proteins. 【Results】 We successfully cloned two odorant binding protein genes from M. alternatus, which were named as MaltOBP2 (GenBank accession no. KP120891) and MaltOBP6 (GenBank accession no. KP120892), with the opening reading frame of 402 bp and 408 bp in length, respectively. Their translated amino acid sequences contain four conserved cysteines, suggesting that they belong to the Minus-C OBP subfamily. MaltOBP2 and MaltOBP6 have six deduced α-helix regions, and the sites of α-helix regions are very similar between each other. However, the type and polarity of their putative ligand binding sites are completely different. Tissue expression profiles showed that MaltOBP2 and MaltOBP6 were differentially expressed in the head, antennae, maxillary (labial) palpi, abdominal apex and legs of M. alternatus adults, with the highest expression level in the head. The expression levels of MaltOBP2 and MaltOBP6 in antennae were not higher than those in other tissues. Developmental expression profiles showed that MaltOBP2 and MaltOBP6 had the highest expression level in the antennae of pupa and the head of larva, respectively. Prokaryotic expression vectors pET32a-MaltOBP2 and pET32a-MaltOBP6 were successfully constructed and the recombinant proteins were successfully expressed in Escherichia coli. Low temperature (16℃ and 20℃) induced higher levels of the expression of recombinant proteins in the supernatant, and long induction time (12 h) increased the expression of the protein. 【Conclusion】 In this study, the full-length cDNA of two Minus-C OBP genes were cloned from M. alternatus. Their encoded proteins have different physiological functions due to their different ligand binding sites. Expression profiling results suggested that these two OBP genes are not only involved in the olfactory recognition of M. alternatus, but also in other physiological functions such as taste sensing and chemical sensing. Our results lay the foundations for studying the structure and function of MaltOBP2 and MaltOBP6, and are also helpful to our understanding of chemical sensing in M. alternatus.