1.      Introduction

Juvenile Hormone (JH) is a sesquiterpenoid secreted by corpora allata in Insects. Juvenile Hormone plays an important role in physiology, development and reproduction in insect lifecycle. The JH titter in insects was regulated by JH biosynthesis and its degradation by specific enzymes [1]. JH levels are dramatically decreased at the final instar of larval growth, where the ecdysone levels are very high and responsible for pupation. The major enzymes involved in reducing and scavenging the JH before moult of last instar larval stage to pupa were Juvenile Hormone Esterase (JHE), Juvenile Hormone Epoxide Hydrolase (JHEH) and Juvenile Hormone Diol Kinase (JHDK). Formation of JH acid metabolite occurs by the hydrolysis of methyl ester of JH by JHE and hydration of epoxide moiety to JH diol is done by JHEH or JHEH acid diol will be formed by the simultaneous action of both the enzymes JHE and JHEH [2, 3].

Most of the research was concentrated on the mechanism and action of JHE in the JH degradation pathway. When compared to the JHE, very little is known about JHEH though it seems to be as critical as JHE in insect development. A microsomal epoxide hydrolase known as JHEH is a hydrolytic enzyme that belongs to the family α/β hydrolase. Due to the irreversible mechanism of JHEH on JH degradation pathway it could be applied to control insect pests [4]. JHEH genes have been characterized in many insects. In lepidopteran insect pests the JHEH genes are isolated from Manduca sexta [5], Ctenocephalides felis [6], Heliothis virescens [7], Trichoplusia ni [8], Spilarctia obliqua [9,10].

The insect pests attacking the silkworms and its host plant has increased and a new pest attack is being identifying regularly. These pests cause severe damage to the silkworm host plant and economic loss of Sericulture industry. Recently leaf defoliators become a serious threat to seri-farmers as they voraciously feed on young leaves of mulberry which are more important for chawki rearing. Among them Diaphania pulverulentalis (Hampson), commonly called as Leaf roller, has been observed as a severe pest on mulberry since 1995 in Karnataka state [11] that causes a major damage to Mulberry tender leaves. Inhibition of JH degradation pathway causes changes in the JH titer leads to disturbances in physiology of Insects. JHEH is one of the enzymes involved in the removal of JH from the insects.

In this paper, the cloning of the active sites and functional regions of JHEH sequence related with metamorphosis of last instar larvae in mulberry leaf webber - Diaphania pulverulentalis is reported. The obtained partial sequence of JHEH gene was analyzed by Insilico studies.

2.      Materials and Methods

2.1.  Insect Rearing

The larvae of Diaphania pulverulentalis of Mulberry were collected from infested mulberry gardens of surrounding villages of Chittoor District, Andhra Pradesh, India and rearing has done under optimum laboratory conditions. The larvae were fed with fresh V-1 variety mulberry tender leaves. They were reared in insect cage under controlled conditions (24+1⁰C, 75+5% RH) and a photoperiod of 16:8 [12]. The cuticles of day 2, 3 and 4 of fifth instar larvae were dissected and preceded for the RNA isolation.

2.2.  RNA isolation

The total RNA was extracted from cuticles of second, third and fourth days of fifth instar larvae of Diaphania pulverulentalis by TRIzol (Invitrogen) method. The tissues were weighed and frozen in liquid nitrogen. The tissue was homogenized with ceramic mortar and pestle.1 ml of TRIzol per 100 mg of tissue was added and left for 30 min in room temperature until the sample (Tissue sample + TRIzol reagent) becomes liquefied. The samples were centrifuged at 12,000 rpm for 10 min at 4⁰C. Later, the supernatant was transferred into new tube and added 0.2ml of chloroform per 1 ml of TRIzol. It was mixed vigorously and centrifuged at 12,000 rpm for 15 min at 4⁰C. The aqueous layer was carefully separated and added 0.5 ml of Isopropanol. It was incubated in -20⁰c for 50 min. The precipitated RNA was collected by centrifugation at 12,000 rpm for 10 min at 4⁰C. The pellet was twice washed with 75% ethanol and allowed it for partial drying. The partial dried RNA pellet was dissolved in 40 µl of DEPC treated water. The quality and quantity of RNA was measured at 260/280 and 260/230 nm absorbance ratios with a spectrophotometer Nano Drop and was viewed by performing 2% Agarose gel electrophoresis (Sigma Low EEO). The RNA samples were stored at -80⁰C.

2.3.  First Strand cDNA synthesis

2 µg of total RNA sample was used to synthesize the single stranded cDNA. Oligo dT primer (50 µM), dNTP mixture (10 µM) and RNase free water were added to the total RNA and made up the volume to 10 µl. It was incubated for 5 min at 65⁰C and was immediately chilled on ice. To the above reaction mixture 5X Prime script buffer, RNase inhibitor (40U/µl), Prime Script Reverse Transcriptase (200U/µl, Clontech) were added and made up to the volume 20µl. The reaction mixture was mixed properly by vigorous shaking and incubated it for 10 min at 30⁰C followed by 60 min at 42⁰C. Finally, the Reverse Transcriptase enzyme was deactivated by incubating at 95⁰C for 5 min. The sample was cooled on ice. Working condition of cDNA was checked by PCR with 18s rRNA (internal control primers). The cDNA samples were stored in -20⁰C.

2.4.  Primer design

The DpJHEH primers were designed based on its homologous sequences from Spodoptera litura, Manduca sexta, Heliothis virescens, Spilarctia obliqua, Helicoverpa armigera and Trichoplusia ni. Primer-Blast and Primer3 tools were used to check the presence of self complementarity, hairpin formation and dimerization.

2.5.  Amplification of DpJHEH sequence

A pair of degenerate primers (148F: 5^’ TNGAYMNTRAHGMGTGGTGGG 3^’, JHEH6R: 5^’ CCAAYWGTRTCAGGYTTSGTRGC 3^’) were used in the amplification of JHEH gene in Diaphania pulverulentalis. The PCR has been run on automated Master cycler Nexus (Applied Biosystems) using Platinum TM PCR super mix high fidelity. The volume of reaction mixture was 50 µl having 45 µl of Platinum TM PCR high fidelity mixture, 0.5 µl each of 200 µM forward primer (148F), reverse primer (JHEH6R), 1µl of cDNA and 3µl of PCR grade water. Gradient PCR was performed. The PCR conditions used were 2 min at 94⁰C, 40 cycles of 15 sec at 94⁰C, 30 sec at 46-51⁰C, 1min at 68⁰C and 2 min at 68⁰C.

The amplified products of expected size were analyzed on 1.5% Agarose gel (Sigma, Low EEO) in 1X Tris-Acetate-EDTA buffer (40 mM Tris, 40 mM acetic acid and 1 mM ethylene diamine tetra acetic acid) stained with Ethidium bromide at 100 V for 60 min. The obtained bands were visualized by using Gel doc system. The generated expected size bands were cut from the gel, purified and eluted by PCR cleanup/DNA gel extraction kit (TAKARA) as per manufacturer’s instructions. The purified products were sequenced by Sangers dideoxy nucleotide sequencing method. The chromatogram output was checked manually for minimizing ambiguities and base calling errors.

2.6.  Insilico analysis of the generated DpJHEH gene

2.6.1.         Translated BLAST (blastx): The obtained nucleotide sequence similarity was determined based on BLAST search using the NCBI BLAST [13] server with comparison made to Non-redundant protein database by using blastx. The results were checked manually with respect to the percentage of identity and low e-values. The Partial sequence of DpJHEH was submitted to NCBI ORF finder.

2.7.  Multiple Sequence Alignment

The nearest homologues of DpJHEH which were obtained in BLASTX results, retrieved from NCBI database are subjected to MAFFT [14] to identify the conserved regions. Further the Motif finder (www.genome.jp/tools/motif/) and NCBI conserved domain search [15] was used to identify the biological significance sequence patterns and conserved domains in the DpJHEH ORF sequence.

2.8.  Phylogenetic Analysis

Phylogenetic relatedness of DpJHEH and JHEH sequences of six lepidopteran insect pests was done by Phylogeny.fr [16] to identify the evolutionary relationship among the JHEH sequences of insect pests. The tree was generated by Neighbour Joining method using Clustal format alignment.

2.9.  Structural analyses

The obtained DpJHEH protein sequence was subjected to PROTPARAM to explain the primary structural features like amino acid composition, Theoretical PI, Molecular weight, Instability index, grand average of hydropathicity.

Secondary structural analysis was done by SOPMA (Self Optimized Prediction Method with Alignment) [17] to identify the percentage of alpha helices, beta turns, extended strands and random coils in DpJHEH protein sequence.

3.      Results

The 2% Agarose gel electrophoresis of total RNA showed high integrity bands of 28s rRNA and 18s rRNA in all the samples. The purity values of total RNA at 260/280 absorbance are within the range of 1.9 to 2.0. The gel picture of Total RNA is shown in the (Figure 1-4).

The examination of cDNA working condition with 18S rRNA internal control primers was showed with well amplified bands with 200bp size. The gel picture was shown in the (Figure 2). The gradient PCR results inferred that expected amplicon size with 800 bps of DpJHEH was amplified at 50⁰C annealing temperature. The PCR gel picture and purified product of PCR was shown in the (Figure 3 & 4) respectively. The amplified partial DpJHEH was shown in (Figure 5).

The partial DpJHEH showed 71% homology with JHEH sequence of Spodoptera litura, 70% identity with Helicoverpa armigera, 69% identity with Spodoptera exigua, 68% with Amyelois transitella, 67% with Papilio xuthus and 66% with Heliothis virescens. The first ten homologues of JHEH gene of Diaphania pulverulentalis based on blastx results were shown in the (Table 1).

The comparison between the homologues of DpJHEH by MAFFT showed the HGWP motif, Epoxide hydrolase N-terminus (EHN-Position 2-99), alpha/beta hydrolase fold (Abhydrolase_1-Position 85-187) and Alpha/beta hydrolase family (Abhydrolase_6-Position 86-175). The multiple sequence alignment was shown in (Figure 7).

Three motifs Epoxide Hydrolase N-terminus (EHN-Position 2-99), alpha/beta hydrolase fold (Abhydrolase_1-Position 85-187) and Alpha/beta hydrolase family (Abhydrolase_6-Position 86-175) were identified in DpJHEH ORF sequence. The results of NCBI conserved domain search showed two functional domains EHN and Abhydrolase_1 super family in translated protein sequence of DpJHEH. The results were showed in (Figure 8A & B).

The generated phylogenetic tree showed that the partial DpJHEH sequence is evolutionarily closer to the JHEH sequence of Amyelois transitella.  The genetic divergence from the common ancestor can be observed in between JHEH sequences of Diaphania and Spodoptera species. The tree view of Phylogeny.fr was as shown in the (Figure 9).

The PROTPARAM output reveals that the DpJHEH contains 244 amino acids, 27822.19 molecular weight, 9.01 theoretical PI, 36.91 instability index, -0.007 GRAVY. The instability index of the sequence is below as 40. Henceforth the DpJHEH protein was predicted as stable. The protein sequence of DpJHEH showed 33.6% of alpha helices, 19.26% extended strands, 9.84% beta turns and 37.30% random coils. The sequence and secondary structure representation (Figure 10A), the graphical representation of secondary structure (Figure 10B) and amino acid composition (Figure 10C) of DpJHEH are shown below.

4.      Discussion

In insect life cycle, degradation of JH by JHE and JHEH plays a major role in JH titre during the time of secretion from Corpora Allata. Because of less information, there is no much research on JHEH. But due to irreversible mechanism on JH degradation pathway, it is very important to study the JHEH. To this point it could be applied to control the insect pests [1, 2, 18-20]. In Lepidoptera it has been suggested that JH acid act as a better substrate for JHEH than JH in the presence of JH binding protein. Until now the cloning studies of JHEH were done in few Lepidopterans namely Manduca sexta [5, 17, 21, 22] Heliothis virescens [7] and Spilarctia obliqua [18]. In the present study we report the cloning of partial JHEH sequence of Diaphania pulverulentalis and its Bioinformatics analysis. Since it is a seriously identified pest on mulberry this is the first reported sequence in NCBI on JHEH gene of Diaphania pulverulentalis. The present research work reveals that the translated sequence of the amplified partial JHEH sequence showed the HGWP motif region which is an oxyanion hole-the most conserved region in all lepidopteran JHEH protein sequences. The high sequence similarity and identity of DpJHEH with its nearest Lepidopterans concludes that these proteins could derive from a same ancestral gene and perform similar biological function. The genetic divergence have also be known and understood among different JHEH sequences of insect pests. The alignment of amino acid sequence of DpJHEH reveals that they share common conserved regions. Insilico studies of partial DpJHEH showed that its amino acid sequence contains three motif and two conserved domains that are mainly involved in catalytic functions. The Phylogenetic analysis revealed that the Amyelois transitella is the evolutionary closer to the DpJHEH. The amino acid composition, theoretical PI, Instability index was calculated. The instability index of the partial DpJHEH is below 40 concluding that the protein is stable. The secondary structural features alpha helix, beta strand, extended strand and coils were identified in DpJHEH.

5.      Conclusion

This is the first report on JHEH partial gene from Diaphania pulverulentalis. Insilico studies were done on the structural and functional features that could be used for further studies to elucidate the physiological and molecular functions. This work makes an important contribution toward understanding the features of JHEH gene in JH metabolism. Prediction of structural and functional features by Insilico studies facilitates the experimental analysis which leads to development of molecular biocontrol strategies like development of a recombinant biopesticides or even epoxide hydrolases could be identified as the best sources for getting used during the Xenobiotic metabolism in detoxifying the poisonous epoxides. The Insilico analysis was pre-requisite in predicting structural and functional features thereby facilitating the experimental analysis.

Table 1: Blastx results against non-redundant protein database for partial DpJHEH gene sequence.

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