بررسی بیان ژن آلفا-آمیلاز گوارشی لاروهای سوسک کلرادوی سیب‌زمینی (Leptinotarsa decemlineata Say) تحت تاثیر دو عصاره‌ پروتئینی گیاهی

Considering the relevance of insect α-amylases and natural α-amylase inhibitors present in plant seeds to protect against insect damage, the purpose of this research was to investigate the effect of white bean and rapeseed protein extracts on the digestive α-amylase gene expression in the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say). For this purpose, preliminary in vitro and in vivo trials were performed to determine the inhibitory activity of seed proteins on the third and fourth instar larvae (L3 and L4) of CPB. The inhibitory effects of white beans and rapeseed protein extracts on the digestive enzyme of L3 were 48% and 44%, and in L4 were 62% and 55%, respectively. The α-amylase activity of L3 fed by leaf treated with white bean and rapeseed proteins was inhibited by 80% and 55%, and the α-amylase activity of L4 was reduced by 74% and 36%, respectively. While the mortality rate in the control was recorded 7%, in the white bean and rapeseed treatments were 67% and 60%, respectively. For investigation of gene expression profile, total RNA was isolated from whole body of L3 and L4. The integrity of RNA was evaluated and converted to cDNA. The specificity of the cDNA amplicon was verified by the single known size bands on agarose gel. The α-amylase gene expression profile was evaluated using real-time quantitative PCR (RT-qPCR) and ribosomal protein S18 as housekeeping gene. Calculated gene expression ratio demonstrated that the gut α-amylase gene of two different larval stages grown on white bean and rapeseed proteins treated leaves had significantly differentiated expression and was up-regulated in L3 and down-regulated in L4 compared to control. The intensity of treatment bands on agarose gel were increased and decreased in L3 and L4 compared to control, respectively. The Rapeseed proteinaceous extract treatments had the highest expression ratio (2.4-fold) at L3 and the lowest expression ratio at L4 (0.54-fold) compared to control. In the White bean proteinaceous extract treatments, a significant increase in expression ratio (2.04-fold) at L3 and non-significant decrease in expression ratio (0.78-fold) at L4 were observed compared to control. Our data suggest that the hyper-production of digestive α-amylase in L3 is elicited to compensate for the inhibition of the enzyme activity at an earlier stage. As well, reduction of α-amylase gene expression suggests the existence of a negative feedback of plant proteinaceous extracts on the last instar larvae. These findings indicate that the white bean and rapeseed proteins induced larval lethality by impaired food intake and regulated digestive α-amylase activity and gene expression in CPB.