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【目的】克隆烟夜蛾Helicoverpa assulta(Guenée)性肽受体基因并分析其表达模式,为深入研究性肽与交配后反应的关系奠定基础。【方法】采用RT-PCR方法,从烟夜蛾雌蛾性信息素腺体中得到性肽受体基因c DNA全序列。利用荧光定量PCR方法,分析该基因的表达模式。【结果】序列分析结果显示,烟夜蛾性肽受体基因c DNA全长2 048 bp,命名为Hass SPR(Gen Bank登录号:AFH53182.1)。该基因的开放阅读框长1 275 bp,编码424个氨基酸残基,序列中含有7个跨膜域结构,预测分子量和等电点分别为48.6 k Da和9.25。序列比对分析表明,Hass SPR与近缘种棉铃虫H.armigera和其他蛾类性肽受体的氨基酸序列一致性分别达98.35%和超过84%,与已经报道的其他昆虫的性肽受体的氨基酸序列一致性也在64%以上。不同组织表达分析表明,Hass SPR在测定的1日龄雌蛾不同组织中均有表达,以在脑中的表达量最高。时序表达分析表明,在羽化前1天至羽化后6日龄雌蛾的信息素腺体中均有表达,以3日龄表达量最高。雌蛾交配后,Hass SPR在性信息素腺体和脑中的表达量显著上调,而在交配囊和卵巢中的表达量显著下调。【结论】从烟夜蛾雌蛾性信息素腺体中克隆得到性肽受体基因Hass SPR,其表达模式提示该基因的表达水平与雌蛾的生殖生理和生殖行为有关。
【Objective】 The objective of this study is to clone the Helicoverpa assulta (Guenée) peptide receptor gene of Helicoverpa assulta and analyze its expression pattern, which lays a foundation for further study on the relationship between the peptide and the post-mating reaction. 【Method】 The full-length cDNA of the sex-peptide receptor gene was obtained from the sex pheromone glands of female moth by RT-PCR. Fluorescence quantitative PCR was used to analyze the expression pattern of this gene. 【Result】 The results of sequence analysis showed that the genomic DNA of H. pylori was 2048 bp in length and named Hass SPR (Gen Bank accession number AFH 53182.1). The open reading frame of this gene is 1 275 bp in length and encodes 424 amino acid residues. It contains seven transmembrane domains and its predicted molecular weight and isoelectric point are 48.6 kDa and 9.25, respectively. Sequence alignment analysis showed that the amino acid sequence identities of Hass SPR and other closely related H. armigera and other moth peptide receptors were 98.35% and 84%, respectively. Compared with other insect peptoid receptors The amino acid sequence consistency is also above 64%. Expression analysis of different tissues showed that Hass SPR was expressed in different tissues of 1-day-old female moths to determine the highest expression in the brain. Time-series analysis showed that the expression of pheromone was observed in female moths 6 days before emergence and 6 days after eclosion, and reached the highest at 3 days. After female moth mating, the expression of Hass SPR in sex pheromone gland and brain was significantly up-regulated, while the expression of Hass SPR was significantly down-regulated in both mating and ovary. 【Conclusion】 The Hass SPR gene was cloned from sex pheromone glands of female moths. The expression pattern of this gene suggests that the expression level of this gene is related to reproductive physiology and reproductive behavior of female moths.