Development associated profiling of chitinase and microRNA of Helicoverpa armigera identified chitinase repressive microRNA

Abstract

Expression of chitinase is developmentally regulated in insects in consonance with their molting process. During the larval-larval metamorphosis in Helicoverpa armigera, chitinase gene expression varies from high to negligible. In the five-day metamorphic course of fifth-instar larvae, chitinase transcript is least abundant on third day and maximal on fifth day. MicroRNA library prepared from these highest and lowest chitinase-expressing larval stages resulted in isolation of several miRNAs. In silico analysis of sequenced miRNAs revealed three miRNAs having sequence similarity to 3'UTR of chitinase. Gene-targeted specific action of these miRNAs, was investigated by luciferase reporter having 3'UTR of chitinase. Only one of three miRNAs, miR-24, inhibited luciferase expression. Further, a day-wise in vivo quantification of miR-24 in fifth-instar larvae revealed a negative correlation with corresponding chitinase transcript abundance. The force-feeding of synthetic miR-24 induced significant morphological aberrations accompanied with arrest of molting. These miR-24 force-fed larvae revealed significantly reduced chitinase transcript abundance.

Figure 1. Transcript abundance of H. armigera chitinase (hachi).

The relative abundance of hachi transcript in 5^th instar larvae of H. armigera (1–5 day old) was determined by Real-time qPCR. Total RNA isolated from the midgut tissue was used as template and normalised to internal control, β-actin. Expression levels of hachi were measured using Comparative C_T method. A non-template control was run with every experiment. Representative data (mean ±S.D.) from three independent experiments is shown.

Figure 2. Nucleotide sequence of 3′UTR of H. armigera chitinase.

A 1057 bp fragment encoding the 3′UTR of hachi has been cloned in pGEM-Te vector and sequenced. The nucleotide sequence of 3′UTR corresponding to miR-2, miR-24 and miR-131 (miRBase) is shown. The bases in black are completely identical while those in red are mismatches.

Figure 3. Functional validation of chitinase-specific miRNAs using luciferase-based reporter assay in Sf21 cells.

The 3′UTR of hachi was cloned downstream to firefly luciferase (Fluc) in pIB vector. Sf21 cells were co-transfected with recombinant pIB-Fluc-3′UTRhachi, pIB-RLuc control vector and miR-2 or miR-131 or miR-24 (10–250 nM). Dual-luciferase reporter assays were performed 48 hrs after transfection. Firefly/Renilla luciferase ratios were calculated. Histogram depicts the normalised luciferase activity from three independent experiments. The following controls were set up: Control 1: Only Sf21 cells; Control 2: (pIB): Sf21cells transfected with pIB vector alone; Control 3: (FLuc+RLuc): Sf21 cells were co-transfected with pIB-Fluc+pIB-Rluc. The control set of experiments were also repeated thrice in triplicates.

Figure 4. Quantitation of miR-24 in H. armigera larvae.

The relative abundance of miR-24 transcript was estimated in 5^th instar larvae of H. armigera (1–5 day old) by Real-time qPCR. Total RNA was extracted from the midgut tissue and used as template. Transcripts were normalised to β-actin control. Expression levels of miR-24 were measured using Comparative C_T method. A non-template control was run with every experiment. Representative data (mean ±S.D.) from three independent experiments is shown.

Figure 5. Phenotypic effect of miR-24 in H. armigera larvae.

The H. armigera larvae (4^th instar, two-day old) were force-fed with synthetic miR-24 (0.02 nmoles) using a microinjector. Larvae fed with Ringer's buffer and scrambled synthetic miR-24 served as controls. The larvae were monitored upto 48 hrs and morphological changes were observed.

Figure 6. Transcript abundance of hachi in miR-24 fed H. armigera larvae.

The H. armigera larvae (4^th instar, two-day old) were force-fed with synthetic miR-24 (0.02 nmoles) and midgut tissue was dissected 24 hrs post-feeding. Total RNA was isolated from the midgut tissue of miR-24 fed (treated), scrambled miR-24 fed (non-specific control) and buffer-fed (control) larvae. Chitinase transcript was examined by Real-time qPCR using β-actin as internal control. Expression levels of hachi were measured using Comparative C_T method. A non-template control was run with every experiment. Representative data (mean ±S.D.) from three independent experiments is shown.