›› 2018, Vol. 61 ›› Issue (5): 537-545.doi: 10.16380/j.kcxb.2018.05.003

• 研究论文 • 上一篇    下一篇


司品法, 周琼*, 崔中翌   

  1. (湖南师范大学生命科学学院, 长沙 410081)
  • 出版日期:2018-05-20 发布日期:2018-05-20

Cloning, prokaryotic expression and tissue expression profiling of an odorant binding protein gene BminOBP25 from Bactrocera minax (Diptera: Tephritidae)

SI Pin-Fa, ZHOU Qiong*, CUI Zhong-Yi   

  1.  (College of Life Sciences, Hunan Normal University, Changsha 410081, China)
  • Online:2018-05-20 Published:2018-05-20

摘要: 【目的】昆虫的气味结合蛋白(odorant binding proteins, OBPs)与嗅觉识别密切相关,在触角感受器淋巴液内运输外界的脂溶性气味分子顺利到达嗅觉受体过程中起着关键的作用。本研究对柑橘大实蝇Bactrocera minax的气味结合蛋白基因进行了克隆和表达分析,旨在更好地了解气味结合蛋白在柑橘大实蝇嗅觉识别中的作用及为进一步研究柑橘大实蝇嗅觉传递的分子机制奠定基础。【方法】利用RT-PCR和RACE技术克隆柑橘大实蝇的气味结合蛋白基因,并进行生物信息学分析;构建重组表达载体pET28a(+)-BminOBP25,转化到大肠杆菌Escherichia coli BL21(DE3)中,SDS-PAGE及Western blotting鉴定重组表达蛋白;采用荧光定量PCR检测该基因在柑橘大实蝇成虫不同组织中的表达。【结果】克隆获得柑橘大实蝇气味结合蛋白基因的cDNA全长序列,命名为BminOBP25(GenBank登录号:MH181875)。测序结果表明,BminOBP25开放阅读框全长447 bp,编码148个氨基酸,预测分子量为17.5 kD,编码序列具有OBPs典型的6个保守半胱氨酸和6个α螺旋区域特征。在IPTG诱导下目标蛋白以6×His标签融合蛋白的形式在宿主菌中得到稳定表达。荧光定量PCR分析表明,BminOBP25 mRNA在成虫触角、头(去除触角)、胸、腹、足、翅和产卵器中均有表达,其中在触角、头(去除触角)、足和产卵器中表达量较高。【结论】BminOBP25在柑橘大实蝇成虫触角、头、足和产卵器中具有高转录活性,提示该基因在非嗅觉组织中可能也具有生理功能,特别是可能在昆虫的取食与产卵地选择过程中起重要作用,其功能还需深入研究。本研究实现了BminOBP25基因的原核表达,为深入研究BminOBP25基因的功能奠定基础。

关键词: 柑橘大实蝇, BminOBP25, 气味结合蛋白, 基因克隆, 组织表达分析, 荧光定量PCR

Abstract: 【Aim】 The odorant binding proteins (OBPs) of insects are closely related to olfactory recognition and play a key role in the successful delivery of fat-soluble odorant molecules in the lymph nodes of the antennal sensory receptors to the olfactory receptors. In order to understand the roles of OBPs in olfactory recognition and to lay foundation for studying the molecular mechanisms of olfactory transmission in Bactrocera minax, an OBP gene was cloned and its expression profiles were analyzed. 【Methods】 The full-length cDNA sequence of OBP gene was cloned from B. minax using RT-PCR and RACE techniques, and subjected to bioinformatics analysis. The recombinant expression vector pET28a(+)-BminOBP25 was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant protein was identified by SDS-PAGE and Western blotting. The expression profiles of OBP gene in different tissues of B. minax adults were detected by quantitative real-time PCR (qPCR). 【Results】 An OBP gene was cloned from B. minax and named BminOBP25 (GenBank accession no.: MH181875). Its ORF is 447 bp in length encoding 148 amino acids with a predicted molecular weight of 17.5 kD. The encoded protein has typical six conserved cysteines and six α-helices. The recombinant expression vector pET28a(+)-BminOBP25 was constructed and the target protein was stably expressed in host bacteria in the form of 6×His tag fusion protein after IPTG induction. qPCR analysis showed that BminOBP25 mRNA was expressed in antennae, head (without antennae), thorax, abdomen, leg, wing and ovipositor of adults, with higher expression levels in antenna, head (without antennae), leg and ovipositor.【Conclusion】 BminOBP25 has high transcriptional activity in antennae, heads, legs and ovipositors of B. minax adults, suggesting that BminOBP25 may also have physiological functions in non-olfactory tissues, and especially may play important roles during the selection of insect feeding and spawning sites. Its functions need further study. In this study, the prokaryotic expression of BminOBP25 was achieved, laying a foundation for further study of its functions.

Key words: Bactrocera minax, BminOBP25, odorant binding protein (OBP), gene cloning, tissue expression profiling, qPCR