亚洲玉米螟卵黄原蛋白基因的克隆、表达谱及对UV-A胁迫的响应

【目的】本研究克隆亚洲玉米螟Ostrinia furnacalis卵黄原蛋白(vitellogenin, Vg)基因,分析其表达模式,旨在探究UV-A胁迫对亚洲玉米螟Vg基因表达及生殖力的影响。【方法】采用RT-PCR和RACE技术克隆亚洲玉米螟Vg基因的全长,并用生物信息学方法分析其特征;利用RT-qPCR检测亚洲玉米螟不同发育阶段(卵、1-5龄幼虫、蛹和成虫)、雌成虫不同组织(头、足、表皮、卵巢、中肠和脂肪体)中和雌成虫在UV-A胁迫不同时间(0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5和4 h)下该基因的相对表达量。测定UV-A照射不同时长(0, 1, 2, 3和4 h/d)后亚洲玉米螟成虫的生殖及F_1代发育情况。【结果】克隆获得亚洲玉米螟Vg基因的序列,命名为OfVg(GenBank登录号:MK782978)。该基因全长5 760 bp,开放阅读框(ORF)长5 331 bp,编码1 776个氨基酸,蛋白相对分子量为202.10 kD,等电点为9.06。OfVg包含Vg-N, DUF 1943D和VWD 3个功能结构域,无跨膜区结构。系统发育分析表明,OfVg与鳞翅目其他昆虫的Vg的亲缘关系最近。发育表达谱表明,OfVg基因在雌成虫中表达量显著高于其他龄期的表达量,且在雌虫羽化24 h时OfVg表达量最高;组织表达谱表明,OfVg基因在雌成虫脂肪体中特异表达。UV-A照射能诱导雌成虫OfVg的表达,随着照射时间的延长,其表达量先下降再快速上升,在照射3.5 h时其表达量最高,然后骤降;UV-A处理1, 2和3 h/d雌成虫的产卵量显著增加,且F_1代幼虫发育历期显著延长。【结论】OfVg基因在亚洲玉米螟不同发育阶段、雌成虫不同组织中和UV-A照射不同时间的雌成虫中差异表达。本研究为深入研究UV-A胁迫对亚洲玉米螟发育和繁殖的影响奠定了基础。

Cloning and expression profiling of vitellogenin gene and its response to UV-A stress inOstrinia furnacalis(Lepidoptera: Crambidae)

【Aim】 This study aims to explore the effects of UV-A stress on the vitellogenin (Vg) gene expression and fecundity of the Asian corn borer Ostrinia furnacalis by cloning Vg and analyzing its expression pattern. 【Methods】 The full-length cDNA of Vg gene was cloned from O. furnacalis with RT-PCR and RACE techniques and analyzed by bioinformatic methods. The relative expression levels of Vg gene in different developmental stages (egg, 1st-5th instar larva, pupa and adult), different tissues (head, leg, cuticle, ovary, midgut and fat body) of female adults, and female adults of O. furnacalis exposed to UV-A for different time (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h) were detected by RT-qPCR. The reproduction and development of the F₁ generation of O. furnacalis adults after exposure to UV-A for 0, 1, 2, 3 and 4 h/d were measured. 【Results】 The complete sequence of Vg gene from O. furnacalis was cloned and named as OfVg (GenBank accession no.: MK782978). Its full-length cDNA is 5 760 bp and contains a 5 332 bp open reading frame (ORF), encoding 1 776 amino acids with the predicted molecular weight of 202.10 kD and isoelectric point of 9.06. OfVg contains three functional domains (Vg-N, DUF 1943D and VWD) but has no transmembrane region. The phylogenetic analysis showed that OfVg was most closely related to Vg proteins of other lepidopteran insects. Developmental expression profiling showed that the expression level of OfVg in female adult was significantly higher than those in other developmental stages, reaching the peak in 24 h-old female adult. Tissue expression profiling revealed that OfVg was specifically expressed in the fat body of female adults. UV-A stress induced the expression of OfVg in female adults, with the expression level decreasing firstly and then increasing with the increase of exposure time, reaching the maximum at 3.5 h post exposure to UV-A, and then decreasing abruptly. When the adults were exposed to UV-A for 1, 2 and 3 h/d, their fecundity increased, and the developmental duration of larvae of F₁ generation was prolonged significantly. 【Conclusion】 The expression level of OfVg varies in different developmental stages, different tissues of female adults and female adults of O. furnacalis exposed to UV-A for different time. This study lays a foundation for further studying the effect of UV-A stress on the development and reproduction of O. furnacalis.