Identification and expression profiling of Helicoverpa armigera microRNAs and their possible role in the regulation of digestive protease genes

• 96 conserved and 90 novel miRNAs were identified in Helicoverpa armigera by deep sequencing.
• Most of the identified miRNAs were insect-specific, and more than 50 miRNAs were Lepidoptera-specific.
• MiRNAs were differentially expressed in CanPI-7 fed larvae as compared to the larvae fed on control diet.
• H. armigera miRNAs were found to have target sites in several protease genes.
• Negative correlation in the relative abundance of miRNAs and their target mRNAs was evident in certain cases.

The present investigation is an effort to determine the possible roles of microRNAs (miRNAs) in the regulation of protease gene expression in Helicoverpa armigera upon exposure to plant protease inhibitors (PIs). Using Illumina platform, deep sequencing of 12 small RNA libraries was performed from H. armigera larvae fed on artificial diet (AD) or recombinant Capsicum annuum PI-7 (rCanPI-7) incorporated diet, at various time intervals (0.5, 2, 6, 12, 24, and 48 h). Sequencing data were analyzed with miRDeep2 software; a total of 186 unique miRNAs were identified from all the 12 libraries, out of which 96 were conserved while 90 were novel. These miRNAs showed all the conserved characteristics of insect miRNAs. Homology analysis revealed that most of the identified miRNAs were insect-specific, and more than 50 miRNAs were Lepidoptera-specific. Several candidate miRNAs (conserved and novel) were differentially expressed in rCanPI-7 fed larvae as compared to the larvae fed on AD. H. armigera miRNAs were found to have target sites in several protease genes as well as in protease regulation related genes such as serine PI and immune reactive PI. As expected, negative correlation in the relative abundance of miRNAs and their target mRNAs was evident from qualitative real time polymerase chain reaction analysis. The investigation revealed potential roles of miRNAs in H. armigera protease gene regulation.

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