GSTs和GSH-Px在家蚕幼虫抵御球胞白僵菌感染过程中的作用

Roles of GSTs and GSH-Px in Denfence of Silkworm Larvae against Beauveria bassiana Infection

本实验研究家蚕幼虫解毒和抗氧化防御体系在抗真菌感染过程尤其是真菌毒素解毒过程中的作用。检测了家蚕幼虫感染球孢白僵菌以及注射球孢白僵菌毒素之后血淋巴、中肠和脂肪体中谷胱甘肽-S-转移酶（GSTs）活力、谷胱甘肽过氧化物酶（GSH—Px）活力、谷胱甘肽还原酶（GR）活力和谷胱甘肽（GSH）含量的变化及GST、GSH-Px基因表达水平的变化，同时还检测了注射GSH和毒素对家蚕幼虫感染球孢白僵菌后存活率的影响。结果表明球孢白僵菌感染家蚕幼虫后脂肪体、中肠、血淋巴中的GSTs、GR、GSH-Px酶活力性显著提高，各组织中BraGSTd1、BmG5Ts1、BmGSTo1和BmGSH-Px的表达量在感染后期（54～72h）也显著上调表达，说明家蚕幼虫可以通过增加酶活力并上调解毒和抗氧化因子的表达量以增强抗真菌感染能力。毒素注射实验表明家蚕幼虫对球孢白僵菌毒素的解毒作用主要在脂肪体和血淋巴中依赖GSTs、GR和GSH来完成，同时定量实验表明BmGSTs1和BmGSTo1在注射毒素24h后显著上调表达，说明它们可能是家蚕幼虫对球孢白僵菌毒素的主要解毒基因。存活率实验结果表明GSH能够缓解球孢白僵菌毒素对蚕体的损害，延长家蚕幼虫的半数致死时间（约6h）。本研究结果表明家蚕幼虫GSTs和GSH-Px构成的解毒和抗氧化防御系统在抗真菌感染过程中起到重要作用，丰富了对家蚕与病原真菌互作机制的认识。

The roles of detoxification and antioxidation system in anti-fungal infection and detoxification of mycotoxins in silkworm larvae were studied in this paper. The variations in GSTs （glutathione-S-transferases）, GSH-Px （glutathione peroxidase） and GR （Glutathione Reductase） activities, GSH （glutathione） content, and expression levels of GSTs and GSH-Px genes in hemolymph, midgut and fat body of silkworm larvae infected with Beauveria bassiana or injected with Beauveria toxins were examined. Moreover, the effects of GSH and Beauveria toxins on the survival rates of silkworm larvae infected with B. bassiana were also examined. The results showed that the enzyme activities of GSTs, GSH-Px and GR and the expression level of BmGSTd1, BmGSTs1, BmGSTo1 and BmGSH-Px genes in fat body, midgut and hemolymph were all significantly increased （especially from 54 hours to 72 hours post infection） against B. bassiana infection; meanwhile, GSH （glutathione） content was significantly decreased, indicating that silkworm larvae could up-regulated the enzyme activities and expression levels of the detoxification and antioxidation factors to enhance the anti-fungal abilities. Toxins injection assay showed that the detoxification in silkworm larvae against Beauveria toxins was mainly depended on GSTs, GR and GSH in fat body and haemolymph. Besides, the results of quantitative real time PCR showed that the expression levels of BmGSTs1 and BmGSTo1 genes were significantly up-regulated at 24 hours after injection with toxins, suggesting that they were the key genes for detoxification in silkworm larvae. Furthermore, the bioassay results showed that GSH could relieve the damage generated by Beauveria toxins and extend the LT50 （lethal time of 50%） about 6 hours. Based on these results, it was confirmed that the detoxification and antioxidation system consisting of GSTs and GSH-Px in silkworm larvae plays an important role in antifungal infection, which offered insights into the molecular mechanism of interaction between the host and fungal pathogens.