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Patnaik, Bharat Bhusan,Hwang, Hee-Ju,Kang, Se Won,Park, So Young,Wang, Tae Hun,Park, Eun Bi,Chung, Jong Min,Song, Dae Kwon,Kim, Changmu,Kim, Soonok,Lee, Jae Bong,Jeong, Heon Cheon,Park, Hong Seog,Han, MDPI 2015 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.16 No.12
<P>The Lycaenidae butterflies, <I>Protantigius superans</I> and <I>Spindasis takanosis</I>, are endangered insects in Korea known for their symbiotic association with ants. However, necessary genomic and transcriptomics data are lacking in these species, limiting conservation efforts. In this study, the <I>P. superans</I> and <I>S. takanosis</I> transcriptomes were deciphered using Illumina HiSeq 2500 sequencing. The <I>P. superans</I> and <I>S. takanosis</I> transcriptome data included a total of 254,340,693 and 245,110,582 clean reads assembled into 159,074 and 170,449 contigs and 107,950 and 121,140 unigenes, respectively. BLASTX hits (<I>E</I>-value of 1.0 × 10<SUP>−5</SUP>) against the known protein databases annotated a total of 46,754 and 51,908 transcripts for <I>P. superans</I> and <I>S. takanosis</I>. Approximately 41.25% and 38.68% of the unigenes for <I>P. superans</I> and <I>S. takanosis</I> found homologous sequences in Protostome DB (PANM-DB). BLAST2GO analysis confirmed 18,611 unigenes representing Gene Ontology (GO) terms and a total of 5259 unigenes assigned to 116 pathways for <I>P. superans</I>. For <I>S. takanosis</I>, a total of 6697 unigenes were assigned to 119 pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Additionally, 382,164 and 390,516 Simple Sequence Repeats (SSRs) were compiled from the unigenes of <I>P. superans</I> and <I>S. takanosis</I>, respectively. This is the first report to record new genes and their utilization for conservation of lycaenid species population and as a reference information for closely related species.</P>
Patnaik, Bharat Bhusan,Kang, Seong Min,Seo, Gi Won,Lee, Hyo Jeong,Patnaik, Hongray Howrelia,Jo, Yong Hun,Tindwa, Hamisi,Lee, Yong Seok,Lee, Bok Luel,Kim, Nam Jung,Bang, In Seok,Han, Yeon Soo Molecular Diversity Preservation International (MD 2013 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.14 No.10
<P>CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, <I>Tenebrio molitor.</I> The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. <I>In silico</I> analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to <I>T. molitor</I> immune response against various microbial pathogens.</P>
Bharat Bhusan Patnaik,Seong Min Kang,Gi Won Seo,Hyo Jeong Lee,Hongray Howrelia Patnaik,Yong Hun Jo,Hamisi Tindwa,Yong Seok Lee,Bok Luel Lee,Nam Jung Kim,In Seok Bang,Yeon Soo Han 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10
CD63, a member of tetraspanin membrane protein family, plays pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic ‘Cys-Cys-Gly’ motif and ‘Cys188’ residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcript was upregulated the maximum 4.5 fold in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also made significant increase in the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.
Patnaik, Bharat Bhusan,Park, So Young,Kang, Se Won,Hwang, Hee-Ju,Wang, Tae Hun,Park, Eun Bi,Chung, Jong Min,Song, Dae Kwon,Kim, Changmu,Kim, Soonok,Lee, Jae Bong,Jeong, Heon Cheon,Park, Hong Seog,Han, Hindawi Publishing Corporation 2016 International journal of genomics Vol.2016 No.-
<P><I>Vespa mandarinia</I> found in the forests of East Asia, including Korea, occupies the highest rank in the arthropod food web within its geographical range. It serves as a source of nutrition in the form of Vespa amino acid mixture and is listed as a threatened species, although no conservation measures have been implemented. Here, we performed<I> de novo</I> assembly of the<I> V. mandarinia</I> transcriptome by Illumina HiSeq 4000 sequencing. Over 60 million raw reads and 59,184,811 clean reads were obtained. After assembly, a total of 66,837 unigenes were clustered, 40,887, 44,455, and 22,390 of which showed homologous matches against the PANM, Unigene, and KOG databases, respectively. A total of 15,675 unigenes were assigned to Gene Ontology terms, and 5,132 unigenes were mapped to 115 KEGG pathways. The zinc finger domain (C2H2-like), serine/threonine/dual specificity protein kinase domain, and RNA recognition motif domain were among the top InterProScan domains predicted for<I> V. mandarinia</I> sequences. Among the unigenes, we identified 534,922 cDNA simple sequence repeats as potential markers. This is the first transcriptomic analysis of the wasp<I> V. mandarinia</I> using Illumina HiSeq 4000. The obtained datasets should promote the search for new genes to understand the physiological attributes of this wasp.</P>
Bharat Bhusan Patnaik,Hongray Howrelia PATNAIK,박기범,조용훈,이용석,한연수 한국곤충학회 2015 Entomological Research Vol.45 No.2
Insect apolipophorin‐III is an exchangeable protein that is abundantly found in the hemolymph, and serves an important role in lipid transport, development, and innate immunity. In this study, we examined the role of apolipophorin‐III (TmapoLp‐III) during the adult eclosion stages of Tenebrio molitor by RNA interference (RNAi) analysis. After silencing of the mRNA transcripts at both larval and pupal stages, adult phenotypic defects were noticed. Defects included the incomplete shedding of pupal skin, shorter extension of the elytra, and improper folding of the hind wings. Most of the adults were malformed and died possibly due to dehydration. We also showed the involvement of TmapoLp‐III in conferring resistance to T. molitor larvae against Listeria monocytogenes infection. Mortality was found to be lower in non‐silenced intoxicated larvae while the TmapoLp‐III silenced larvae showed a significant susceptibility after 7 days post‐injection with a dose of 106 cfu/larvae.
Hamisi Tindwa,Bharat Bhusan Patnaik,Dong Hyun Kim,Seulgi Mun,Yong Hun Jo,Bok Ruel Lee,Yong Seok Lee,Nam Jung Kim,In Seok Bang,Yeon Soo Han 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10
Peptidoglycan recognition proteins (PGRPs) are family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (IMD) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts followed by a challenge with L. monocytogenes showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infections in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.
Ju Young Noh,Bharat Bhusan Patnaik,Hamisi Tindwa,Gi Won Seo,Dong Hyun Kim,Hongray Howrelia Patnaik,Yong Hun Jo,Yong Seok Lee,Bok Ruel Lee,Nam Jung Kim,In Seok Bang,Yeon Soo Han 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10
Apolipophorin III (apoLp-III) is a well-known hemolymph protein having a functional role in lipid transport and immune response of insects. We cloned full-length cDNA encoding putative apoLp-III from larvae of the coleopteran beetle, Tenebrio molitor (TmapoLp-III), by identification of clones corresponding to the partial sequence of TmapoLp-III, subsequently followed with full length sequencing by a clone-by-clone primer walking method. The complete cDNA consists of 890 nucleotides, including an ORF encoding 196 amino acid residues. Excluding a putative signal peptide of the first 20 amino acid residues, the 176-residue mature apoLp-III has a calculated molecular mass of 19,146 Da. Genomic sequence analysis with respect to its cDNA showed that TmapoLp-III was organized into four exons interrupted by three introns. Several immune-related transcription factor binding sites were discovered in the putative 5’-flanking region. BLAST and phylogenetic analysis reveals that TmapoLp-III has high sequence identity (88%) with Tribolium castaneum apoLp-III but shares little sequence homologies (<26%) with other apoLp-IIIs. Homology modeling of Tm apoLp-III shows a bundle of five amphipathic helices, including a short helix 3’. The ‘helix-short helix-helix’ motif was predicted to be implicated in lipid binding interactions, through reversible conformational changes and accommodating the hydrophobic residues to the exterior for stability. Highest level of TmapoLp-III mRNA was detected at late pupal stages, albeit it is expressed in the larval and adult stages at lower levels. The tissue specific expression of the transcripts showed significantly higher numbers in larval fat body and adult integument. In addition, TmapoLp-III mRNA was found to be highly up-regulated in late stages of L. monocytogenes or E. coli challenge. These results indicate that TmapoLp-III may play an important role in innate immune responses against bacterial pathogens in T. molitor.
Dong Hyun Kim,Bharat Bhusan Patnaik,Gi Won Seo,Seong Min Kang,Yong Seok Lee,Bok Luel Lee,In Seok Bang,Yeon Soo Han 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10
We have identified novel ricin-type (R-type) lectin by sequencing of random clones from cDNA library of the coleopteran beetle, T.molitor. The cDNA sequence is comprised of 495 bp encoding a protein of 164 amino acid residues and shows 49% identity with galectin of Tribolium castaneum. Bioinformatics analysis shows that the amino acid residues from 35 to 162 belong to ricin-type β-trefoil structure. The transcript was significantly upregulated after early hours of injection with peptidoglycans derived from Gram (+) and Gram (-) bacteria, beta-1, 3 glucan from fungi and an intracellular pathogen, L. monocytogenes suggesting putative function in innate immunity.
Yong Hun Jo,Bharat Bhusan Patnaik,Se Won Kang,Seunghan Oh,Dong Hyun Kim,Mi Young Noh,Gi Won Seo,Heon Cheon Jeong,Ju Young Noh,Hee Ju Hwang,Ji-Eun Jeong,Yeon Soo Han,Yong Seok Lee 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10
Most traditional genome sequencing projects involving infectious viruses include culturing and purification of the virus. This can present difficulties as an analysis of multiple populations from multiple locations may be required to acquire sufficient amount of high-quality DNA for sequence analysis. The electrophoretic method provides a strategy whereby the genomic DNA sequences of the Korean isolate of Pieris rapae granulovirus (PiraGV-K) were analyzed by purifying it from host DNA by pulsed-field gel electrophoresis, thus simplifying sampling and labor time. The genomic DNA of infected P. rapae was embedded in agarose plugs, digested with a restriction nuclease and methylase, and pulsed-field gel electrophoresis (PFGE) was used to separate PiraGV-K DNA from the DNA of P. rapae, followed by mapping of fosmid clones of the separated viral DNA. The double-stranded circular genome of PiraGV-K encodes 120 open reading frames (ORFs), covering 92% of the sequenced genome. BLAST and ORF arrangement showed the presence of 78 homologs to other genes in the database. The mean overall amino acid identity of PiraGV-K ORFs was highest with the Chinese isolate of PiraGV (~99%), followed up with Choristoneura occidentalis ORFs at 58%. PiraGV-K ORFs were grouped, according to function, into 10 genes involved in transcription, 11 involved in replication, 25 structural protein genes, and 15 auxiliary genes. Genes for Chitinase (ORF 10) and cathepsin (ORF11), involved in the liquefaction of the host, were found in the genome. The recovery of PiraGV-K DNA genome by pulse-field electrophoretic separation from host genomic DNA had several advantages, compared with its isolation from particles harvested as virions or inclusions from the P. rapae host. We have sequenced and analyzed the 108,658 bp PiraGV-K genome purified by the pulsed field electrophoretic method. The method appears to be applicable to the analysis of genomes of large viruses. The chitinase, identified by PiraGV-K genome sequence, was functionally characterized by quantitative PCR, Western blot analysis, immunohistochemistry and transmission electron microscopy.