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为阐明热激蛋白90(heat shock protein 90,HSP90)在甜菜夜蛾Spodoptera exigua(Hbner)幼虫抵抗高温过程中的作用,克隆了其HSP90基因cDNA全长序列,并检测了在系列高温胁迫下不同龄期幼虫体内其相对表达量。根据已报道的热激蛋白90基因序列同源性设计简并引物,利用反转录聚合酶链式反应(RT-PCR)结合cDNA末端快速扩增(RACE)技术成功克隆了甜菜夜蛾HSP90基因全长cDNA(GenBank登录号FJ862050)。该cDNA序列开放阅读框长2154bp,编码717个氨基酸,预测的相对分子量和等电点分别为82.6kD和5.0。该序列具有HSP90家族的典型特征和特殊的功能结构域,并且与多种生物的HSP90基因序列有较高的同源性。为了研究HSP90抵抗高温的作用,构建荧光定量RT-PCR体系,检测了37,39,41,43和45℃胁迫下甜菜夜蛾不同龄期幼虫体内HSP90表达量的变化。结果表明,高温胁迫对甜菜夜蛾幼虫体内的HSP90表达具有明显的诱导作用。幼虫体内HSP90表达量随着温度升高呈增加的趋势。43℃和45℃胁迫下,各龄幼虫体内HSP90的表达量均显著高于常温(P<0.05),但不同龄期之间没有显著差异。这说明HSP90在甜菜夜蛾幼虫抗高温中起到重要作用。
In order to elucidate the role of heat shock protein 90 (HSP90) in the resistance of Spodoptera exigua (Hbner) larvae to high temperature, we cloned the full-length cDNA of HSP90 gene and examined the effects of heat shock protein 90 Under different age larvae in vivo relative expression. The degenerate primers were designed according to the reported sequence homology of heat shock protein 90 and the HSP90 gene was successfully cloned by RT-PCR and rapid amplification of cDNA ends (RACE) Full-length cDNA (GenBank Accession No. FJ862050). The open reading frame of this cDNA sequence was 2154bp, encoding 717 amino acids. The predicted relative molecular weights and isoelectric points were 82.6kD and 5.0, respectively. The sequence has typical features and special functional domains of HSP90 family, and has high homology with HSP90 gene sequences of many organisms. In order to study the effect of HSP90 on high temperature resistance, a real-time fluorescent quantitative RT-PCR system was constructed to detect the expression of HSP90 in different instar larvae of beet armyworm at 37, 39, 41, 43 and 45 ℃. The results showed that high temperature stress induced the expression of HSP90 in beet armyworm larvae significantly. The expression of HSP90 in larvae tended to increase with increasing temperature. Under the stress of 43 ℃ and 45 ℃, the expression of HSP90 in all instar larvae was significantly higher than normal temperature (P <0.05), but there was no significant difference between different age groups. This suggests that HSP90 plays an important role in the resistance of beet armyworm larvae to high temperatures.