Insect growth regulators (IGRs) are insecticides that disrupt the normal development of target insects. Among the IGR insecticides, juvenile hormone (JH)-related IGRs are of particular interest because they stimulate or interfere with the formation of JH receptor complex. In the precious studies, novel JH-related IGRs with JH agonist (JHA) and antagonist (JHAN) activity were identified by using yeast two-hybrid system transformed with the Aedes aegypti JH receptor complex. In this study, the transcriptomic responses of Aedes albopictus were investigated upon JHA and JHAN, respectively. These results will provide important information about understanding of impact of JH-related IGRs in transcription level.

Bacillus thuringiensis (Bt) is a gram-positive bacterium that produces parasporal crystal proteins known as endotoxins or Cry proteins. The Cry protoxins are then cleaved by insect midgut proteinases to form active Bt toxins. The activated Cry protein then binds to specific receptors at the midgut epithelium. Cadherin-like and aminopeptidase N (APN) proteins are involved in Bt toxin binding by interacting sequentially with different toxin structures. Aminopeptidase N (APNs) from several insect species have been shown to be putative receptors for these toxins. We have characterized four different midgut APNs(APN1, APN2, APN3, APN4) cDNAs from S. exigua. Forward primers and reverse primers for confirmation of four different midgut APNs were designed based on their sequences cloned from the cDNA libraries. Quantitative RT-PCR procedures includes 42℃ for 20min (cDNA synthesis), 99℃ for 5min, and 35 cycles (94℃ for 1min, and 60℃ for 50 s) for collection. Four aminopeptidase N isoforms were confirmed with qRT-PCR. Sequence analysis was performed by BlastX search the National Center for Biotechnology Information(NCBI) nucleotide. Furthermore, double-stranded RNAs(dsRNAs) were synthesized. DsRNAs were determined for bioassay.

Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures and these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, and mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with Polh gene at the N-terminus including an adaptor and enterokinase (EK) site between Polh and EGFP was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells according to three steps; cell harvest, sonication and EK digestion. Through the final enterokinase digestion, EGFP was presented mainly in the supernatant (93.3%) and the supernatant also showed a pure EGFP band. These results suggest that the combined procedure of Polh fusion expression and enterokinase digestion can used for the rapid and easy purification of other proteins.

The GA application on grapevines induces parthenocarpy, fruit set without fertilization, and the inhibition of pollen tube growth. But the molecular mechanism underlying this inhibition is not understood. Similar defective pollen tube growth within the transmitting tract has been reported in the mutant of GABA transaminase (GABA-T), referred to as pollen-pistil-interaction2 (pop2) in Arabidopsis. In spite of the similarity of pollen tube growth inhibition observed in GA-applied grapevines with that of pop2, only the effects of GABA on stress responses in grapevines have been reported. In present study, transcriptional changes of Vitis GABA metabolic genes, together with changes in GABA levels with or without GA application were analyzed to define how GA application restrained the pollen tube growth in grapevines. A GA solution (Dongbu, Seoul, Korea) at 100 ppm was onto inflorescence clusters 14 days before full bloom (DBF) and clusters were harvested at 0, 1, 2, 4, 7, 9, 12, 14, 16, and 19 days after GA application. Harvested inflorescence samples were immediately frozen in LN2 and extracted RNA and amino acid. The GABA contents were analyzed using high-performance liquid chromatography (Agilent 1100 HPLC, Agilent Technologies, Inc., Santa Clara, USA) equipped with a C18 column (4.6 mm×150 mm, 3.5 μm/VDS optilab, Berlin, Germany), according to the manufacturer’s instructions. Without GA application, the simultaneous high expressions of VvGAD1, VvGAD4 and VvGABA-T2 during 10 to 5 days before full bloom (DBF) showing the activation of GABA metabolism. But the contents of GABA were low before 2 DBF, and it peaked only at near full bloom when expression levels of VvGABA-T2 remained low. After GA application, the contents of GABA were constant during 10 to 5 DBF, although transcription levels of both VvGAD1 and VvGABA-T2 rapidly declined less than 30% of the levels observed without GA application. However, the GABA levels increased more than 2-fold only at near full bloom, compared to those without GA application, and at that time, expression levels of VvGAD1 up-regulated more than 3-fold and those of VvGABA-T2 kept low. But other amino acid contents did not show significant changes. In case of VvSSAHDs, their transcriptional changes with or without GA application were not correlated with GABA levels. These results indicates that GABA levels before pollination is tightly regulated, but GA application alters the GABA-shunt to accumulate excess GABA more than needed for proper pollen tube growth at full bloom. Gibberellin application alters the GABA-shunt to accumulate excess GABA resulting in inhibition pollen tube growth in grapevines.