关键词: 苹果蠹蛾, 耐热性, 热激蛋白, 系统发育, 热激诱导, 相对表达模式

Abstract: The codling moth, Cydia pomonella (Lepidoptera: Tortricidae), is a worldwide quarantine pest and possesses the plasticity in thermotolerance ability. The temperature fluctuations may enhance its tolerance to heat stress. In the present study, we determined the critical threshold of tolerance to high temperature by bioassay method in C. pomonella laboratory population, and applied the techniques of homology-based cloning, RACE and real-time quantitative PCR (RT-qPCR) to elucidate the function of Hsp90 responding to heat stress in C. pomonella. The results of bioassay showed that the mortality of C. pomonella was significantly increased as temperature was elevated and time prolonged. When the 1st-5th instar larvae were exposed to 50℃ and 52℃ for 2, 5 and 10 min, the 3rd instar larvae were found to be the most sensitive and the 5th instar larvae the most resistant. The 1st-4th instar larvae were killed 100% when exposed to 50℃ for 10 min and 52℃ for 5 min, while the survival rate of 5th instar larvae still maintained at 25.0% and 11.1%, respectively. The full-length cDNA of hsp90 gene was obtained from the 5th instar larvae of C. pomonella exposed to 35℃, which is 2 470 bp in length and has an open reading frame (ORF) of 2 148 bp encoding a protein of 716 amino acids with the deduced molecular weight of 82.07 kDa. This gene was registered in GenBank under the accession number JN624775 and designated as Cphsp90. CpHsp90 shares 96% amino acid sequence identity with the Hsp90 from Ostrinia furnacalis and Mamestra brassicae, suggesting that the Hsp90 family is highly conserved. The result of RT-qPCR showed that the Cphsp90 was heat inducible, and the relative expression level of Cphsp90 mRNA was positively correlated with the heat stress when the larvae were exposed to 32-44℃ for 1 h. When the larvae of C. pomonella were exposed to 35℃, Cphsp90 was expressed abundantly in cuticle with the expression level significantly higher than that in the hemolymph, fat body and midgut. The result of the mRNA expression pattern of the Cphsp90 under higher temperature after pretreatment at 35℃ for 3 h indicated that the maximum expression level in the larvae preheated was induced at higher temperature (45℃ for 10 min) compared with the larvae not preheated (40℃ for 10 min). This result further proved that preheat treatment enhanced the thermotolerance of 5th instar larvae. It is so concluded that the positive expression of the Cphsp90 mRNA may play an important role in theromotolerance and the plasticity of C. pomonella.

Key words: Cydia pomonella, heat resistance, heat shock protein (Hsp), phylogeny, heat induction, relative expression pattern