梨小食心虫普通气味受体基因GmolOR20的克隆及表达分析
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摘要
【目的】通过克隆梨小食心虫Grapholita molesta触角中的普通气味受体(odorant receptor, OR)OR20基因,明确其在不同发育期及成虫不同组织中的表达特征,为进一步研究其功能提供理论依据。【方法】根据梨小食心虫雌虫触角转录组数据,利用RT-PCR克隆梨小食心虫OR20基因的完整开放阅读框;采用qRT-PCR检测该基因在不同发育期(卵、1-5龄幼虫、蛹和雌雄成虫)、成虫不同组织(触角、去除触角的头、胸、腹、足、翅)以及不同日龄(1, 3, 5和7日龄)成虫触角中的表达量。【结果】克隆获得梨小食心虫GmolOR20基因cDNA序列(GenBank登录号:MH898864)。该基因完整开放阅读框为1 284 bp,编码427个氨基酸,预测蛋白分子量为49.83 kD,理论等电点为8.57,具有7个跨膜结构域。序列比对和系统进化树结果表明,梨小食心虫GmolOR20与苹果蠹蛾Cydia pomonella CpomOR15和豆荚小卷蛾Cydia nigricana CnigOR15亲缘关系较近,氨基酸序列一致性分别为87%和84%。发育表达模式结果显示,GmolOR20在不同发育时期均有表达,雌雄成虫中的表达量显著高于其他发育期的表达量(P<0.05),但雌、雄虫间的表达量差异不显著。组织表达模式结果表明,GmolOR20主要在成虫触角中高丰度表达,且雌虫触角中的表达量极显著高于雄虫触角中的表达量(P<0.01);GmolOR20在不同日龄成虫的触角中均有表达,且在1和3日龄成虫触角中的表达水平显著高于其他日龄(P<0.05)。【结论】根据GmolOR20基因的表达谱分析结果,推测GmolOR20可能参与梨小食心虫对植物挥发物和性信息素的识别。
【Aim】 The objective of this study is to clone the gene of a general odorant receptor(OR) OR20 of the oriental fruit moth, Grapholita molesta, and to investigate its expression profiles in different developmental stages and adult tissues of the moth, so as to provide a theoretical basis for further functional study of the odorant receptor gene. 【Methods】 According to the antennal transcriptome of female adults of G. molesta, the open reading frame(ORF) of OR20 gene of G. molesta was cloned by PCR. The expression levels of this gene at different developmental stages(egg, 1-5 instar larva, pupa, and female and male adults), in different adult tissues(antenna, head with antenna removed, thorax, abdomen, leg and wing), and in antennae of adults of different age(1, 3, 5 and 7 day-old) were detected by qRT-PCR. 【Results】 The cDNA sequence of GmolOR20 gene(GenBank accession no.: MH898864) in G. molesta was obtained. Its complete ORF is 1 284 bp in length, encoding 427 amino acids with the predicted molecular weight of 49.83 kD, the isoelectric point of 8.57, and seven transmembrane domains. Multiple sequence alignment and phylogenetic tree analyses revealed that GmolOR20 is closely related with CpomOR15 of Cydia pomonella and CnigOR15 of Cydia nigricana, with the amino acid sequence identity of 87% and 84%, respectively. Developmental stage-specific expression results revealed that GmolOR20 was expressed in various developmental stages of G. molesta, and its expression levels in female and male adults were significantly higher than those in other developmental stages(P<0.05), but showed no significant difference between female and male adults. Tissue expression profiles revealed that GmolOR20 was highly expressed in the antenna of adults, with extremely significantly higher expression level in the antenna of female adult than in the antenna of male adult(P<0.01). GmolOR20 was also expressed in antennae of adults of different day-old, with significantly higher expression levels in antennae of the 1 and 3 day-old adults than in antennae of other day-old adults(P<0.05). 【Conclusion】 Based on the profiling results of GmolOR20, we infer that GmolOR20 may be involved in the recognition of plant volatiles and pheromones in G. molesta.
【Aim】 The objective of this study is to clone the gene of a general odorant receptor(OR) OR20 of the oriental fruit moth, Grapholita molesta, and to investigate its expression profiles in different developmental stages and adult tissues of the moth, so as to provide a theoretical basis for further functional study of the odorant receptor gene. 【Methods】 According to the antennal transcriptome of female adults of G. molesta, the open reading frame(ORF) of OR20 gene of G. molesta was cloned by PCR. The expression levels of this gene at different developmental stages(egg, 1-5 instar larva, pupa, and female and male adults), in different adult tissues(antenna, head with antenna removed, thorax, abdomen, leg and wing), and in antennae of adults of different age(1, 3, 5 and 7 day-old) were detected by qRT-PCR. 【Results】 The cDNA sequence of GmolOR20 gene(GenBank accession no.: MH898864) in G. molesta was obtained. Its complete ORF is 1 284 bp in length, encoding 427 amino acids with the predicted molecular weight of 49.83 kD, the isoelectric point of 8.57, and seven transmembrane domains. Multiple sequence alignment and phylogenetic tree analyses revealed that GmolOR20 is closely related with CpomOR15 of Cydia pomonella and CnigOR15 of Cydia nigricana, with the amino acid sequence identity of 87% and 84%, respectively. Developmental stage-specific expression results revealed that GmolOR20 was expressed in various developmental stages of G. molesta, and its expression levels in female and male adults were significantly higher than those in other developmental stages(P<0.05), but showed no significant difference between female and male adults. Tissue expression profiles revealed that GmolOR20 was highly expressed in the antenna of adults, with extremely significantly higher expression level in the antenna of female adult than in the antenna of male adult(P<0.01). GmolOR20 was also expressed in antennae of adults of different day-old, with significantly higher expression levels in antennae of the 1 and 3 day-old adults than in antennae of other day-old adults(P<0.05). 【Conclusion】 Based on the profiling results of GmolOR20, we infer that GmolOR20 may be involved in the recognition of plant volatiles and pheromones in G. molesta.
引文
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