棉铃虫离子型受体基因HarmIR8a的克隆及表达定位

Cloning and expression localization of ionotropic receptor gene HarmIR8a in Helicoverpa armigera (Lepidoptera: Noctuidae)

【目的】昆虫离子型受体(ionotropic receptors, IRs)是新发现的一类化学感觉受体,对昆虫感受环境中酸类和胺类等物质发挥着重要作用。基因克隆、序列比对以及表达定位的研究将有助于初步解析棉铃虫Helicoverpa armigera离子型受体的功能。【方法】本研究在棉铃虫触角转录组测序和分析的基础上,利用PCR技术克隆了一个离子型受体基因的全长序列,并进行序列结构预测等分析;利用荧光定量PCR技术对该基因在棉铃虫雌、雄成虫不同组织中的表达量进行了分析;同时利用原位杂交技术检测了该基因在棉铃虫雄虫触角中的表达定位。【结果】克隆获得棉铃虫HarmIR8a基因全长cDNA序列(GenBank登录号:MH638313),开放阅读框为2 688 bp,编码895个氨基酸,预测有3个跨膜区域。HarmIR8a氨基酸序列包含了一个氨基末端区域(amino terminal domain, ATD),一个由S1和S2两部分构成的配体结合域(ligand binding domain, LBD),一个孔环(pore loop, P),3个跨膜区(M1, M2和M3)和一个胞内C末端(C terminus)。荧光定量PCR结果显示,HarmIR8a在棉铃虫雌、雄成虫头(除去附器)和触角等组织中均有表达,而且在触角中高表达。棉铃虫雄虫触角原位杂交结果表明,HarmIR8a主要在触角腔锥形感器下表达。【结论】本研究初步探析了HarmIR8a基因的转录水平和表达部位,为进一步研究棉铃虫离子型受体基因的功能和生理作用奠定了基础。

【Aim】 Ionotropic receptors （IRs） are newly discovered chemosensory receptors in insects, which play an important role in insects in sensing acids and amines in the environment. The studies of cloning, sequence alignment and expression localization of genes of IRs will help their preliminary functional analysis in Helicoverpa armigera . 【Methods】 Based on the results of antennal transcriptome analysis, the full-length sequence of an ionotropic receptor gene of H. armigera was cloned by PCR, and its sequence structure was analyzed. The relative expression levels of this gene in different tissues of female and male adults of H. armigera were analyzed by real-time fluorescent quantitative PCR assay, and its expression localization in male antennae was detected by in situ hybridization. 【Results】 The full-length cDNA sequence of HarmIR8a gene （GenBank accession no.： MH638313） was cloned from H. armigera . The HarmIR8a sequence comprises a 2 688 bp ORF and encodes a protein of 895 amino acids with three predicted transmembrane domains. HarmIR8a contains one amino-terminal domain （ATD）, one ligand binding domain （LBD） consisting of two lobes （S1 and S2）, one pore loop （P）, three transmembrane domains （M1, M2 and M3） and one C-terminus. The qPCR results revealed that HarmIR8a was mainly expressed in the head （with appendages removed） and antennae of female and male adults of H. armigera , with the highest relative expression level in the antennae. The results of in situ hybridization of the male adult antennae of H. armigera showed that HarmIR8a was mainly expressed in the antennal coeloconic sensilla. 【Conclusion】 The transcriptional level and expression localization of HarmIR8a were preliminarily determined. This study lays a foundation for further functional characterization and physiological activities of ionotropic receptor genes in H. armigera .