Analysis of cysteine peptidase gene expression during insect developmental stages by RT-qPCR

Leaf-cutting ants are considered the most important herbivorous insects, commonly depending on fresh plant leaves, which serve as substrate for symbiotic fungus culture1. These insects are known for their high damage power to agriculture and natural forests or reforestation. In insect cysteine peptidases have been expressed on stage of development in several organisms, such enzymes have been identified in metamorphosis and tissue reconstruction2, which suggesting these enzymes have a great potential to be used as molecular targets to developing new insecticides. Previously studies in our laboratory, the DNA of a cysteine peptidase of Atta Sexdens ants was cloned,
and recombinant enzyme was expressed in E.coli, purified and tested against cysteine peptidase inhibitors. To understanding the role of the enzyme in these organisms, trying to infer which metabolic pathway is involved, the aim of this present work was analyzed the gene expression quantitatively by RT-qPCR in the different stages of A. sexdens life (pupae, larvae and worker) and in some parts of the worker body (head, mesosoma, gaster and the body without gaster (WWG). The cDNA from insect developmental stages and its body parts were used as a template for independent qPCR reactions. RT-qPCR was performed using the StepOneTM Real-Time PCR System (Thermo Fisher Scientific) with total volume reaction of 12 μL: 6 μl of Power SYBR® Green PCR Master Mix (Thermo Fisher Scientific), 3 μl (about 1.7 ng) of diluted cDNA and 3 μl of mix solution containing forward and reverse primers (according with the F/R relation). Thermal cycling conditions for all genes were: 15 minutes at 95 °C, followed by 40 cycles of 95 °C for 15 seconds, 60 °C for 60 seconds and 95 °C for 15 seconds. An analysis of relative gene expression data was calculated using the 2−ΔΔCt method as described 3 The statistical analyses were performed with GraphPad Prism 5 software. The mean comparisons were computed by analysis of variance (ANOVA). Tukey's multiple comparisons tests were used for post hoc analysis. P < 0.01 (**) and p < 0.001 (***) were considered statistically significant. The gene was expressed throughout the life cycle of the insect which suggests a more general role for the enzyme, as well as the lysosomal function. Among the body parts of adult , there is no difference in the expression pattern gene transcription and the results showed that the gaster represent a significative importance in the cysteine peptidase transcript expression.