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

【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6289 bp,开放阅读框(ORF)5490 bp,编码1829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。

[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,1 st-5 th 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 6289 bp in length with a 5490 bp open reading frame(ORF)encoding a 205.27 ku protein with 1829 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.