亚洲玉米螟卵黄原蛋白受体基因的克隆及在UV-A胁迫下的表达分析
Cloning of the vitellogenin receptor gene and its expression under UV-A stress in Ostrinia furnacalis
刘 芳；孟建玉；苏 丽；张长禹
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DOI：10.7679/j.issn.2095-1353.2021.010
作者单位：1. 贵州大学昆虫研究所，贵州省山地农业病虫害重点实验室，贵阳 550025；2. 贵州省烟草科学研究院，贵阳 550081
中文关键词：亚洲玉米螟；卵黄原蛋白受体；UV-A胁迫；RT-qPCR
英文关键词：Ostrinia furnacalis; vitellogenin receptor; UV-A stress; RT-qPCR
中文摘要：
【目的】 本文旨在克隆亚洲玉米螟Ostrinia furnacalis卵黄原蛋白受体（VgR）基因，分析其表达模式，探索UV-A胁迫对亚洲玉米螟VgR基因表达的影响。【方法】 利用RT-PCR与RACE技术克隆亚洲玉米螟VgR基因的全长序列；运用生物信息学方法分析该基因特征；采用RT-qPCR技术检测不同发育阶段（卵、1-5龄幼虫、蛹、成虫）、雌成虫不同组织（头、足、表皮、卵巢、中肠、脂肪体）、雌成虫不同时长（0、0.5、1、1.5、2、2.5、3、3.5、4和4.5 h）UV-A胁迫下该基因的相对表达量。【结果】 克隆获得了亚洲玉米螟VgR基因，命名为OfVgR（GenBank登录号: MN058042），其全长6 289 bp，开放阅读框（ORF）5 490 bp，编码1 829个氨基酸，预测蛋白分子量为205.27 ku，N端前31个氨基酸为信号肽。序列分析显示，OfVgR具有2个配体结合域（LBD）、2个表皮生长因子前体同源域（EGFP）、跨膜域（TMD）和胞质尾域。系统发育树分析表明，OfVgR与鳞翅目昆虫VgR聚为一支，亲缘关系较近。RT-qPCR检测结果表明，OfVgR在亚洲玉米螟各发育阶段均有表达，其中在卵和雌成虫中高表达，并在雌成虫羽化24 h时表达量达到最高；雌成虫不同组织中，OfVgR在卵巢中表达量最高；OfVgR表达量随着UV-A照射时间的延长呈先下降后上升再下降的趋势，在3.0 h达到最高值。【结论】 亚洲玉米螟OfVgR在不同发育阶段，雌成虫不同组织和UV-A胁迫不同时间的雌成虫中差异表达，对探索UV-A胁迫对亚洲玉米螟生殖的分子机制影响奠定基础。
英文摘要：
[Objectives] To explore the effects of UV-A stress on the expression of the vitellogenin receptor (VgR) gene in the Asian corn borer Ostrinia furnacalis. [Methods] The full-length sequence of the VgR gene was cloned from O. furnacalis with reverse transcription PCR（RT-PCR）and the rapid amplification of cDNA ends（RACE）technique and its characteristics analyzed using bioinformatics methods. Real-time quantitative PCR (RT-qPCR) technology was used to detect the expression of the VgR gene in different developmental stages (eggs, 1st-5th instar larvae, pupae and adults), different tissues of female adults (head, foot, cuticle, ovary, midgut and fat body), and in female adults exposed to UV-A for different periods of time (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 and 4.5 h). [Results] The complete VgR gene was cloned from O. furnacalis and named OfVgR (GenBank login number: MN058042). Its full-length cDNA was 6 289 bp in length with a 5 490 bp open reading frame (ORF) encoding a 205.27 ku protein with 1 829 amino acids, including a putative 31-amino-acid signal peptide at the N-terminus. OfVgR contains conserved domains, including two ligand-binding domains (LBD), two EGF-precursor homology domains (EGFP), a transmembrane domain (TMD) and a cytoplasmic domain. A phylogenic tree indicates that the protein is closely related to the VgR proteins of other Lepidoptera. RT-qPCR results revealed that OfVgR is expressed in all developmental stages of O. furnacalis, with higher expression in eggs and female adults. Expression was highest in female adults 24 h after eclosion. Expression of OfVgR in different adult female tissues was highest in ovary. OfVgR expression first decreased, increased, then decreased with increased duration of UV-A exposure, with peak expression occurring after 3.0 h exposure. [Conclusion] Expression of OfVgR differs in different developmental stages and adult female tissues and expression in female adults is affected by the duration of exposure to UV-A. These findings lay a foundation for investigating the molecular effects of UV-A stress on the reproduction of O. furnacalis.