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posted Mar 4, 2011, 7:14 AM by rajesh gk

Title

Purification and characterization of a protease degrading 30 kDa yolk proteins of the silkworm, Bombyx mori.

Authors

Maki N, Yamashita O.

Laboratory of Sericulture and Entomoresouces, School of Agricultural Sciences, Nagoya University, Japan.

Journal

Insect Biochem Mol Biol. 1997 Aug-Sep;27(8-9):721-8.

Abstract

The second major yolk proteins, 30 kDa proteins (30kPs) of the silkworm, Bombyx mori, which have been provided during oogenesis, are kept continuously unused during embryogenesis and are utilized just before larval hatching. The crude extracts of newly hatched larvae cleaved 30kPs in an in vitro incubation system. A protease was highly purified from newly hatched larvae using ammonium sulfate precipitation, gel filtration and ionic exchange chromatography, and non-denaturing-polyacrylamide gel electrophoresis (ND-PAGE). The protease shared the NH2-terminal amino acid sequence conserved in many serine proteases, and the apparent molecular mass was estimated to be approximately 600 kDa by gel filtration column chromatography. The enzymatic activity was strongly inhibited by elastatinal and diisopropyl fluorophosphate (DFP), indicating that this protease is an elastase-like serine protease. The protease selectively hydrolysed 30kP-1 and 30kP-4 between Ser6 and Ala7, but could not attack other 30kPs such as 30kP-2, 30kP-3 and 30kP-5. Consequently, the protease characterized in the present study is a unique protease which may be specialized for the selective degradation of yolk proteins in silkworm eggs.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/9443372 PMID: 9443372 [PubMed - indexed for MEDLINE]

 

Title

Cloning and expression of a novel gene encoding a new antibacterial peptide from silkworm, Bombyx mori.

Authors

Kim SH, Park BS, Yun EY, Je YH, Woo SD, Kang SW, Kim KY, Kang SK.

National Sericulture & Entomology Research Institute, RDA, Suwon, Korea.

Journal

Biochem Biophys Res Commun. 1998 May 19;246(2):388-92.

Abstract

We differentially screened a novel gene encoding a new antibacterial peptide from the immunized Bombyx mori cDNA library. The gene showed a similar structure to that of cecropin-family, encoding 59 amino acids including a putative leader peptide and mature peptide. The deduced peptide, named Enbocin, had conserved amino acid residues which have been known to play an important role in the antibacterial activities. Enbocin genomic sequence revealed that the transcription unit of Enbocin gene was about 1.2 kb, and the coding sequence was interrupted by an intron of 660 bases. Recombinant Enbocin, expressed under the control of the baculovirus polyhedrin promoter, demonstrated a broad range of antibacterial activities against gram positive and gram negative bacteria.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/9610369 PMID: 9610369 [PubMed - indexed for MEDLINE]

 

Title

cDNA cloning and mRNA expression of L-3,4-dihydroxyphenylalanine decarboxylase gene homologue from the silkworm, Bombyx mori.

Authors

Hwang JS, Kang SW, Goo TW, Yun EY, Lee JS, Kwon OY, Chun T, Suzuki Y, Fujiwara H.

Department of Sericulture and Entomology, National Institute of Agricultural Science and Technology, RDA, Suwon 441-100, South Korea. hwangjs@rda.go.kr

Journal

Biotechnol Lett. 2003 Jun;25(12):997-1002.

Abstract

L-3,4-Dihydroxyphenylalanine decarboxylase (DDC) cDNA, from Bombyx mori that contains an open reading frame of 1437 bp encoding 478 amino acids, was cloned and characterized. Expression analyses of B. mori DDC mRNA by Northern and in situ hybridization indicated that expression of silkworm DDC expression is possibly controlled by neuropeptide hormones in tissue- and stage-specific manners.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/12889838 PMID: 12889838 [PubMed - indexed for MEDLINE]

 

Title

Transcription and Expression of Major VP Gene of Bombyx Mori Parvo-like Virus

Authors

Jinsong Cheng ; Qin Yao ; Meng Lv ; Chen Sun ; Yuanqing He ; Keping Chen

Journal

International Journal of Biology, 2009; 1(2)

Abstract

The Bombyx mori parvo-like virus (China Zhenjiang isolate), termed as BmDNV-Z, replicates only in host’s midgut<br />columnar cells and causes flacherie disease. The viral genome is composed of two sets of different single-stranded<br />linear DNA molecules, Viral DNA 1(VD1) and Viral DNA 2(VD2). Here we cloned the major VP gene from the<br />plasmid contained viral DNA fragments. The gene consists of 1500 nucleotides and the deduced protein has 499 amino<br />acid residues, with the predicted molecular weight of 54934.24Da, isoelectric point of 6.73. The coding sequence was<br />inserted into expression plasmid vector pET-30a and expressed in Escherichia coli BL21 (DE3) under the inducible<br />promoter LacZ. The expressed product was detected by Western blotting with anti-His antibody. Interestingly, there are<br />four laddered band instead of one. We speculated that this virus may use leaky scanning mechanism to express four<br />proteins in one ORF. And we investigated the major VP gene transcriptional time phase in susceptible silkworm through<br />real time quantitative PCR (qPCR). The result showed that the mRNA level of VP gene increased greatly after 24 hours<br />post inoculation

Citation

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Title

Expression of Cocoonase in Silkworm (Bombyx mori) Cells by Using a Recombinant Baculovirus and Its Bioactivity Assay

Authors

Jianjun Yang ; Wenbing Wang ; Bing Li ; Yudan Wu ; Huiling Wu ; Weide Shen

Journal

International Journal of Biology, 2009; 1(2)

Abstract

In this research, the cocoonase gene was cloned by RT-PCR as an 860 bp fragment, including the signal peptide and the<br />core sequence of cocoonase gene. In order to investigate the function of signal peptide, recombinant transfer vector<br />pBacPAK8-Cocoonase-EGFP were constructed by fusing with enhanced green fluorescent protein (EGFP) gene to<br />observe under fluorescence microscope. The purified pBacPAK8-Cocoonase-EGFP DNA was co-transfected with<br />linear virus Bm-BacPAK6 DNA into BmN cells. The homologous recombination occurred in the cells and then the<br />recombinant virus Bm-BacPAK8-Cocoonase-EGFP was obtained. BmN cell was infected with the recombinant virus<br />Bm-BacPAK8-Cocoonase-EGFP, and fluorescent signal was detected in most of the cells under fluorescence<br />microscope at 72 hrs postinfection. Then BmN cells were harvested. Both SDS-PAGE and Western-blotting analysis<br />indicated that the cocoonase was expressed successfully in silkworm (Bombyx mori) baculovirus expression vector<br />system. Furthermore, referred to Astrup methods,used fibrin plate process confirmed that expression product in vitro<br />had cellulolytic activity. We conclude that silkworm expression system can be used successfully to express functional<br />cocoonase.

Citation

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Title

Characterization and Prokaryotic expression of Glucuronyltransferase-S Gene in Silkworm Bombyx mori

Authors

Yukun Chen ; KePing Chen ; Fang Bao ; Qin Yao

Journal

International Journal of Biology, 2009; 1(1)

Abstract

As genome of B. mori is available in GenBank, identification of novel genes of B. mori can be carried out. In this study,<br />we used the in silico cloning method to obtain the Glucuronyltransferase-S (GlcAT-S) gene of B. mori and analysed<br />with bioinformatics tools. The result was confirmed by RT-PCR,prokaryotic expression and western blot. The GlcAT-S<br />cDNA contains a 843bp ORF and has three exons. The deduced protein has 280 amino acid residues, with the predicted<br />molecular weight of 31842. 02 Da, isoelectric point of 9.16, and contains conserved GlcAT domains. The protein shows<br />high degrees of identity with that of some homologous protein from other species.

Citation

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Title

Cloning and Characterization of NAD-dependent Deacetylase Sirtuin 2 Homolog from the Silkworm, Bombyx mori.

Authors

Yijia Li ; Keping Chen ; Qin Yao ; Lu Gao ; Jun Li ; Lin Wang

Journal

International Journal of Biology, 2009; 1(1)

Abstract

Sirtuin2 (Sirt2) is a kind of NAD+-dependent deacetylases ranging from bacteria to human and play an important role in<br />many biological processes especially in lifespan. We performed genome analysis and protein prediction of Sirt2 of B.<br />mori (BmSirt2). The cDNA sequence of BmSirt2 contains an ORF of 1164 bp encoding 387 amino acid residues with a<br />predicted molecular mass and isoelectric point of 43.37 kDa and 5.02, respectively. This protein shows high degrees of<br />identity with other species. Phylogenetic relationship analysis showed that the BmSirt2 protein was in the same<br />subgroup as the Sirt2 from invertebrate animals. RT-PCR analysis of gene expression in multiple tissues showed that<br />Sirt2 gene was widely expressed in B. mori. BmSirt2 was successfully expressed in E. coli with a molecular mass of<br />48.0 kDa. The identification of the recombinant protein by MALDI-TOF-MS and western blotting showed this fusion<br />protein was the correct one.

Citation

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Title

Molecular cloning and characterization of a cDNA encoding a transferrin homolog from Bombyxmori.

Authors

Yun EY, Kang SW, Hwang JS, Goo TW, Kim SH, Jin BR, Kwon OY, Kim KY.

Department of Sericulture and Entomology, National Institute of Agricultural Science and Technology, RDA, Suwon, Korea.

Journal

Biol Chem. 1999 Dec;380(12):1455-9.

Abstract

We isolated a cDNA representing a message that was strongly induced by injection with E. coli in Bombyxmori. The 2160 bpcDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa. The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding. We suggest that the B. mori transferrin plays an important role in the self-defense system.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/10661875 PMID: 10661875 [PubMed - indexed for MEDLINE]

 

Title

The 30kP protease A responsible for 30-kDa yolk protein degradation of the silkworm, Bombyxmori: cDNA structure, developmental change and regulation by feeding.

Authors

Maki N, Yamashita O.

Laboratory of Sericulture and Entomoresouces, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, 464-8601, Nagoya, Japan.

Journal

Insect BiochemMol Biol. 2001 Mar 15;31(4-5):407-13.

Abstract

We have cloned and sequenced the cDNA encoding the major component (43-kDa peptide) of 30kP protease A which selectively hydrolyzes 30-kDa yolk proteins of the silkworm, Bombixmori. The deduced amino acid sequence consisted of 318 amino acids and shared sequences conserved in many serine proteases. Northern blot analysis using the cDNA as probe revealed that 43-kDa peptide mRNA began to rise at the last phase of embryogenesis and reached a maximum level at larval hatching. This level was maintained with some fluctuations throughout post-embryonic development. The concentration of 43-kDa peptide increased greatly toward larval hatching coinciding with the changing pattern of mRNA. When larvae were fed, the peptide concentration abruptly decreased and remained near zero throughout post-embryonic development. The decrease in peptide concentration did not occur, however, when the hatched larvae were starved. Thus, the nutritional shift from endogenous yolk to exogenous food plays a key role in 30kP protease A elimination from neonate larvae.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/11222950 PMID: 11222950 [PubMed - indexed for MEDLINE]

 

Title

Molecular distinction amongst varieties of mulberry using RAPD and DAMD profiles.Free PMC Article

Authors

Bhattacharya E, Ranade SA.

PMB Division, National Botanical Research Institute, RanaPratapMarg, Lucknow 226001 (UP) India. esha_b@rediffmail.com

Journal

BMC Plant Biol. 2001;1:3. Epub 2001 Dec 13.

Abstract

BACKGROUND: Mulberry trees are the most important host for rearing mulberry silkworms in sericulture. Improved varieties of mulberry tree have been developed through traditional breeding procedures. Not much work, however, has been carried out on the molecular characterization of these varieties. Random Amplified Polymorphic DNA (RAPD) and Directed Amplification of Minisatellite DNA (DAMD) methods based on Polymerase Chain Reaction are important tools to analyze genetic diversity of mulberries. These have been used to determine variation amongst nine varieties of Morus spp. maintained at Banthra Research Station of National Botanical Research Institute, Lucknow. RESULTS AND DISCUSSION: The varieties were analyzed using 23 arbitrary sequence decamer primers for RAPD, and 3 minisatellite core sequence primers for DAMD reactions. The RAPD and DAMD band data, (a total of 200 bands), were used to determine the pair wise distances according to Jaccard's algorithm. From these distance values Neighbour Joining (NJ) analyses were carried out separately for the RAPD and the DAMD data. The triploid varieties were found to be most similar to each other using RAPD analysis, while the varieties S13 and S34 were more similar using DAMD analysis. Nearly 85% of the RAPD bands and 91% of the DAMD bands were polymorphic across the nine varieties. CONCLUSIONS: The mulberry varieties could be distinguished by their RAPD and DAMD profiles. As many as five RAPD primers and one DAMD primer generated profiles that can together differentiate all the nine varieties in terms of unique bands.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/11801190 PMID: 11801190 [PubMed - indexed for MEDLINE]PMCID: PMC64612

 

Title

Systematic cloning and analysis of autophagy-related genes from the silkworm Bombyx mori

Authors

Zhang Xuan ; Hu Zhan-Ying ; Li Wei-Fang ; Li Qing-Rong ; Deng Xiao-Juan ; Yang Wan-Ying ; Cao Yang ; Zhou Cong-Zhao

Journal

BMC Molecular Biology (2009), 10 (1) 50

Abstract

Background: Through the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated.

Results: Based on the autophagy pathway in several model organisms and a series of bioinformatic analyses, we have found more than 20 autophagy-related genes from the current database of the silkworm Bombyx mori. These genes could be further classified into the signal transduction pathway and two ubiquitin-like pathways. Using the mRNA extracted from the silkgland, we cloned the full length cDNA fragments of some key genes via reverse transcription PCR and 3' rapid amplification of cDNA ends (RACE). In addition, we found that the transcription levels of two indicator genes BmATG8 and BmATG12 in the silkgland tend to be increased from 1st to 8th day of the fifth instar larvae.

Conclusion: Bioinformatics in combination with RT-PCR enable us to remodel a preliminary pathway of autophagy in the silkworm. Amplification and cloning of most autophagy-related genes from the silkgland indicated autophagy is indeed an activated process. Furthermore, the time-course transcriptional profiles of BmATG8 and BmATG12 revealed that both genes are up-regulated along the maturation of the silkgland during the fifth instar. These findings suggest that the autophagy should play an important role in Bombyx mori silkgland.

Citation

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Biotechnology

posted Jul 9, 2010, 8:48 AM by rajesh gk   [ updated Mar 4, 2011, 8:07 AM ]

Title

[DNA fingerprinting analysis of silkworm embryo cell lines]

[Article in Chinese]

Authors

Pan MH, Feng ZY, Tian ZQ, Liu M, Lu C.

Key Sericultural Laboratory of Agriculture Ministry, College of Sericulture and Biotechnology, Southwest University, Chongqing.

Journal

Fen Zi Xi Bao Sheng Wu Xue Bao. 2006 Dec;39(6):537-43.

Abstract

DNA extraction and the polymerase chain reaction (PCR) were used on DNA genomes study of cell lines of Bombyx mori. DNA polymorphic marker analysis was conducted and DNA fingerprint of cell lines of Bombyx mori. was carried out using ISSR and RAPD. Primers that can reliably find polymorphic bands were screened out. 26 ISSR primers were selected from them any available, and 797 polymorphic bands were abtained through PCR amplification in 9 samples, including 3 embryo cell lines of Bombyx mori (BmE-SWU1, BmE-SWU2, BmE-SWU3), 5 passage cell lines (BmE, BmN, Sf9, Sf21, Hi5) and the embryos from which BmE-SWU1 originated. The ration of polymorphic bands was 89.9%. 43 RAPD primers were selected out through PCR amplification, and 1205 polymorphic bands were obtained in 9 samples. The ration of polymorphic bands was 76.6%. There were many DNA polymorphic bands differences in the cell lines of Bombyx mori. The special DNA markers of the 3 embryo cell lines were found respectively. The similarity index Nei and genetic distance of the 9 samples were calculated and the phylogeny tree of 9 samples was constructed by UPGMA. Results showed that 2 groups were divided,one group including the 3 embryo cell lines and the embryo of XQ has close relative. Another group constructed by five insect cell lines came from different species, their genetic distance was closer than the 3 embryo cell lines.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/17348206 PMID: 17348206 [PubMed - indexed for MEDLINE]

 

Title

Analysis on frequency and density of microsatellites in coding sequences of several eukaryotic genomes.

Authors

Li B, Xia Q, Lu C, Zhou Z, Xiang Z.

The Key Sericultural Laboratory of Agricultural Ministry, College of Sericulture and Biotechnology, Southwest Agricultural University, Chongqing 400716, China.

Journal

Genomics Proteomics Bioinformatics. 2004 Feb;2(1):24-31.

Abstract

Microsatellites or simple sequence repeats (SSRs) have been found in most organisms during the last decade. Since large-scale sequences are being generated, especially those that can be used to search for microsatellites, the development of these markers is getting more convenient. Keeping SSRs in viewing the importance of the application, available CDS (coding sequences) or ESTs (expressed sequence tags) of some eukaryotic species were used to study the frequency and density of various types of microsatellites. On the basis of surveying CDS or EST sequences amounting to 66.6 Mb in silkworm, 37.2 Mb in fly, 20.8 Mb in mosquito, 60.0 Mb in mouse, 34.9 Mb in zebrafish and 33.5 Mb in Caenorhabditis elegans, the frequency of SSRs was 1/1.00 Kb in silkworm, 1/0.77 Kb in fly, 1/1.03 Kb in mosquito, 1/1.21 Kb in mouse, 1/1.25 Kb in zebrafish and 1/1.38 Kb in C. elegans. The overall average SSR frequency of these species is 1/1.07 Kb. Hexanucleotide repeats (64.5%-76.6%) are the most abundant class of SSR in the investigated species, followed by trimeric, dimeric, tetrameric, monomeric and pentameric repeats. Furthermore, the A-rich repeats are predominant in each type of SSRs, whereas G-rich repeats are rare in the coding regions.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/15629040 PMID: 15629040 [PubMed - indexed for MEDLINE]

 

Title

Membrane-penetrating trehalase from silkworm Bombyx mori. Molecular cloning and localization in larval midgut.

Authors

Mitsumasu K, Azuma M, Niimi T, Yamashita O, Yaginuma T.

Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Japan.

Journal

Insect Mol Biol. 2005 Oct;14(5):501-8.

Abstract

The main blood sugar in insects, trehalose, differs from glucose in mammals. To incorporate trehalose into cells and utilize it, tissue cells possess the enzyme trehalase (EC3.2.1.28), which catalyses trehalose into glucose, in the organellar membrane or in the cytoplasm. Soluble and membrane-bound trehalase proteins have been isolated from insects. To date, however, only genes encoding the soluble trehalase have been reported in insects. Soluble trehalase is therefore believed to become localized on the cell surface via modification. In contrast, cDNAs encoding trehalase localized on the apical cell surface via the glycosylphosphatidylinositol-anchor have been isolated from mammalian small intestines. The amino acid sequence contains a specific hydrophobic region and an upstream omega site, which is cleaved for glycosylphosphatidylinositol-attachment, at the C-terminus. Here, we describe a cDNA from the silkworm Bombyx mori that encodes a novel trehalase (type-2) with one transmembrane domain and lacking the omega site. Immunoblotting and immunohistochemical analyses demonstrated that in the midgut tissue of Bombyx larvae, soluble trehalase-1 is present mainly in goblet cell cavities, but membrane-bound trehalase-2 is predominantly seen on the visceral muscle surrounding the midgut. To our knowledge, this is the first report of a cDNA encoding trehalase that penetrates the cell membrane in insects and its cellular localization.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/16164606 PMID: 16164606 [PubMed - indexed for MEDLINE]

 

Title

Endoplasmic reticulum stress response of Bombyx mori calreticulin.

Authors

Goo TW, Park S, Jin BR, Yun EY, Kim I, Nho SK, Kang SW, Kwon OY.

Department of Sericulture and Entomology, National Institute of Agricultural Science and Technology, Suwon, 441-744, Korea.

Journal

Mol Biol Rep. 2005 Sep;32(3):133-9.

Abstract

We isolated a calreticulin cDNA from the silkworm, Bombyx mori. The cDNA encodes 398 amino acid residues of B. mori calreticulin, with an endoplasmic reticulum retentional HDEL motif at its C-terminus and a predicted molecular mass of 45,801 Da. The B. mori calreticulin shows high protein homology with calreticulin from G. mellonella (88%), A. aegypti (71%), D. melanogaster (69%) and H. sapiens (63%). The highest level of mRNA expression of B. mori calreticulin was exhibited in the fat body of this insect. Although expression of B. mori calreticulin was affected by disturbances in intracellular calcium levels, other ER stress conditions such as inhibition of intracellular protein transport, reduction of disulfide formation, glycosylation inhibition, heat shock and oxidative stress did not disrupt induction of B. mori calreticulin.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/16172913 PMID: 16172913 [PubMed - indexed for MEDLINE]

 

Title

Molecular analysis of divergence in tachinid Uzi (Exorista sorbillans) populations in India.

Authors

Chatterjee SN, Taraphdar T, Mohandas TP.

SeriBiotech Laboratory, Central Silk Board, Kodathi Campus, Sarjapur Road, Carmelram, 560035 Bangalore, Karnataka, India. shankerc99@yahoo.com

Journal

Genetica. 2005 Sep;125(1):1-15.

Abstract

Exorista sorbillans is a tachinid endoparasitoid of silkworm, Bombyx mori, and is globally known as uzi. It causes economic injury to the cocoon crop in silkworm cultivating areas of India, except those above 400 m above mean sea level (AMSL) in the foothills of the Himalayas (Darjeeling). It is reported that the sericulture tract of south India became infected with this pest only since 1980 through an accidental transportation of cocoons from West Bengal. To ascertain whether the genome of this parasitoid is differentiating into discrete gene pools in contrasting geo-climatic conditions, molecular profiling of four populations (Es (Annatapur), Es(Ramanagaram), Es (Channapatna) and Es(Kodathi) from south India and Es(Murshidabad) from Murshidabad, West Bengal was undertaken with 13 ISSR, 3 RAPD and six non-random primers designed from various repeat sequences of B. mori . MANOVA indicated significance for the Roy's largest root estimate (55.4; F =18.47; p = 0.002) for the variability contributed by the replication. Further, hierarchical clustering done on the basis of Euclidean distance matrix and Nei's unbiased Phylip clustering put Es(Murshidabad) at the maximum distance from those of south India and 29 markers could also be identified which significantly differentiateEs(Murshidabad) from others. However, Nei's statistics for gene diversity in sub-populations reveal considerably high gene-flow (3.44 and 2.51) among the populations around Bangalore. The gene-flow between Es(Murshidabad) and other population is lowest but cannot be ignored. The comparison of endosymbiont specific 16SrRNA and fts Z gene (partial) sequences through clustalW (gcgMSF) revealed a closer relationship of Es(Murshidabad) with Es(Annatapur) and Es (Ramanagaram) and is not congruent with the relationships discussed above. The significance of this maiden study with a tachinid fly-pest is discussed in the context of understanding the diversification of Uzi fly-pest and also establishing this pest as a relevant biological material for studying microevolution in future.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/16175450 PMID: 16175450 [PubMed - indexed for MEDLINE]

 

Title

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein from the Chinese oak Silkmoth, Antheraea pernyi.

Authors

Kim BY, Park NS, Jin BR, Lee SM.

 

Department of Sericulture and Entomology, Miryang National University, South Korea.

Journal

DNA Seq. 2005 Oct;16(5):397-401.

Abstract

In our research to identify gene involved in the cuticle protein, we cloned a novel cuticle protein gene, ApCP13, from the Chinese oak silkmoth, Antheraea pernyi, larvae cDNA library. The ApCP13 gene encodes a 120 amino acid polypeptide with a predicted molecular mass of 13 kDa and a pI of 4.01, and is intron-less gene. The ApCP13 contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the ApCP13 cDNA is most homologous to another wild silkmoth, A. yamamai CP12 (86% protein sequence identity), followed by Bombyx mori LCP18 (35% protein sequence identity). Northern blot analysis revealed that the ApCP13 showed the epidermis-specific expression. This is the first report of cuticle protein gene in the wild silkmoth, A. pernyi.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/16323268 PMID: 16323268 [PubMed - indexed for MEDLINE]

 

Title

DNA fingerprinting using AFLP markers to search for markers associated with yield attributes in the silkworm, Bombyx mori.

Authors

Gaviria DA, Aguilar E, Serrano HJ, Alegria AH.

 

Center for Molecular Biology and Biotechnology, Universidad Tecnológica de Pereira, Risaralda, Colombia. duberney82@netscape.net

Journal

J Insect Sci. 2006;6:1-10.

Abstract

This study was carried out on 11 Chinese and 12 Japanese silkworm strains maintained by the Center for the Technological Development of Sericulture (CDTS) germplasm bank, located in Pereira, Colombia. The goals were to determine the genetic population structure of the two groups and the association between molecular markers (AFLPs) and important productivity characters. Group analysis showed the separation of the strains according to their geographic origin. The molecular markers and the productivity characters were correlated by multiple variance analysis. The analysis permitted the identification of molecular markers associated with the cocoon weight or the shell weight separately. Some markers were associated with both characters.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/16502509 PMID: 19537986 [PubMed - indexed for MEDLINE]

 

Title

Mulberry improvements via plastid transformation and tissue culture engineering.

Authors

Umate P.

 

Department of Botany, Kakatiya University, Warangal, India. pavan_umate@rediffmail.com.

Journal

Plant Signal Behav. 2010 Jul 9;5(7). [Epub ahead of print]

Abstract

The in vitro tissue culture and micropropagation studies for Morus spp., a pivotal sericulture plant, are well established. The rapid and reproducible in vitro response to plant growth regulator treatments has emerged as an essential complement of transformation studies for this plant species. A major area of study is the use of protoplast culture and fusion techniques where advantages to mulberry improvement can be applied. The advancements in genetic transformation of mulberry are reviewed, and a section on strategy for transforming plastids (chloroplasts) of mulberry is included. A role for mulberry in "molecular farming" is envisioned. The conclusions and future prospects for improvement of this economically important tree species are proposed.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/20495352 PMID: 20495352 [PubMed - as supplied by publisher]

 

Title

Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs.

Authors

Moribe Y, Oka K, Niimi T, Yamashita O, Yaginuma T.

 

Sericulture & Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.

Journal

J Insect Physiol. 2010 Apr 10. [Epub ahead of print]

Abstract

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 degrees C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 degrees C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 degrees C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 degrees C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 degrees C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui. Copyright © 2010 Elsevier Ltd. All rights reserved.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/20371249 PMID: 20371249 [PubMed - as supplied by publisher]

 

Title

Smt3 is required for the immune response of silkworm, Bombyx mori.

Authors

Xu HP, Hao W, He D, Xu YS.

 

Institute of Sericulture & Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou 310029, PR China.

Journal

Biochimie. 2010 Jun 16. [Epub ahead of print]

Abstract

SUMO works in a similar way as ubiquitin to alter the biological properties of a target protein by conjugation. The homologous gene of SUMO named BmSmt3 was identified for the first time in silkworm. The expression of BmSmt3 was enhanced in the fat body of silkworm after immune challenge. However, the expression of BmSmt3 after immune challenge was almost invariant in silk gland, which is the nonimmune organ in silkworm. In addition, the expression of BmRelA and CecropinB1 was decreased significantly in pupae after the BmSmt3 was knocked down in vivo. According to our results, BmSmt3 might participate in the immune response through regulating the expression of BmRelA gene, which can further regulate the expression of antibacterial peptide subsequently in silkworm. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/20561559 PMID: 20561559 [PubMed - as supplied by publisher]

 

Title

Mulberry improvements via plastid transformation and tissue culture engineering.

Authors

Umate P.

 

Department of Botany, Kakatiya University, Warangal, India. pavan_umate@rediffmail.com.

Journal

Plant Signal Behav. 2010 Jul 9;5(7). [Epub ahead of print]

Abstract

The in vitro tissue culture and micropropagation studies for Morus spp., a pivotal sericulture plant, are well established. The rapid and reproducible in vitro response to plant growth regulator treatments has emerged as an essential complement of transformation studies for this plant species. A major area of study is the use of protoplast culture and fusion techniques where advantages to mulberry improvement can be applied. The advancements in genetic transformation of mulberry are reviewed, and a section on strategy for transforming plastids (chloroplasts) of mulberry is included. A role for mulberry in "molecular farming" is envisioned. The conclusions and future prospects for improvement of this economically important tree species are proposed.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/20495352 PMID: 20495352 [PubMed - as supplied by publisher]

 

Title

Smt3 is required for the immune response of silkworm, Bombyx mori.

Authors

Xu HP, Hao W, He D, Xu YS.

 

Institute of Sericulture & Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou 310029, PR China.

Journal

Biochimie. 2010 Jun 16. [Epub ahead of print]

Abstract

SUMO works in a similar way as ubiquitin to alter the biological properties of a target protein by conjugation. The homologous gene of SUMO named BmSmt3 was identified for the first time in silkworm. The expression of BmSmt3 was enhanced in the fat body of silkworm after immune challenge. However, the expression of BmSmt3 after immune challenge was almost invariant in silk gland, which is the nonimmune organ in silkworm. In addition, the expression of BmRelA and CecropinB1 was decreased significantly in pupae after the BmSmt3 was knocked down in vivo. According to our results, BmSmt3 might participate in the immune response through regulating the expression of BmRelA gene, which can further regulate the expression of antibacterial peptide subsequently in silkworm. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Citation

http://www.ncbi.nlm.nih.gov/pubmed/20561559 PMID: 20561559 [PubMed - as supplied by publisher]

posted Jul 5, 2010, 1:50 AM by rajesh gk

Title

A helitron-like transposon superfamily from l epidoptera disrupts (GAAA)(n) microsatellites and is responsible for flanking sequence similarity within a microsatellite family.

Authors

Coates BS, Sumerford DV, Hellmich RL, Lewis LC.

 

USDA-ARS, Iowa State University, Ames, 50011, USA. brad.coates@ars.usda.gov

Journal

J Mol Evol. 2010 Mar;70(3):275-88. Epub 2010 Mar 9.

Abstract

Transposable elements (TEs) are mobile DNA regions that alter host genome structure and gene expression. A novel 588 bp non-autonomous high copy number TE in the Ostrinia nubilalis genome has features in common with miniature inverted-repeat transposable elements (MITEs): high A + T content (62.3%), lack of internal protein coding sequence, and secondary structure consisting of subterminal inverted repeats (SIRs). The O. nubilalis TE has inserted at (GAAA)(n) microsatellite loci, and was named the microsatellite-associated interspersed nuclear element (MINE-1). Non-autonomous MINE-1 superfamily members also were identified downstream of (GAAA)(n) microsatellites within Bombyx mori and Pectinophora gossypiella genomes. Of 316 (GAAA)(n) microsatellites from the B. mori whole genome sequence, 201 (63.6%) have associated autonomous or non-autonomous MINE-1 elements. Autonomous B. mori MINE-1s a encode a helicase and endonuclease domain RepHel-like protein (BMHELp1) indicating their classification as Helitron-like transposons and were renamed Helitron1_BM. Transposition of MINE-1 members in Lepidoptera has resulted in the disruption of (GAAA)(n) microsatellite loci, has impacted the application of microsatellite-based genetic markers, and suggests genome sequence that flanks TT/AA dinucleotides may be required for target site recognition by RepHel endonuclease domains.

Citation

PMID: 20217059 [PubMed - indexed for MEDLINE]

 

Title

Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides.

Authors

Sakamoto S, Pongkitwitoon B, Nakamura S, Maenaka K, Tanaka H, Morimoto S.

 

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

Journal

J Biochem. 2010 Jun 30. [Epub ahead of print]

Abstract

A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence (HMSS) to accelerate secretion of the recombinant GRe-scFv into the hemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the hemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg per liter of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming refolding when it expressed in Escherichia coli.

Citation

PMID: 20592135 [PubMed - as supplied by publisher]

 

Title

Synchronized expression of two caspase family genes, ice-2 and ice-5, in hydrogen peroxide-induced cells of the silkworm, Bombyx mori.

Authors

Sun Y, Wang W, Li B, Wu Y, Wu H, Shen W.

 

Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China. ying_sun@163.com

Journal

J Insect Sci. 2010;10:43.

Abstract

Caspase family proteins play important roles in different stages of the apoptotic pathway. To date, however, functions of Bombyx mori L. (Lepidoptera: Bombycidae) caspase family genes are poorly known. This paper focuses on the morphology, mitochondrial membrane potential, and expression profiles of two novel B. mori caspase family genes (ice-2 and ice-5) in 3 microM hydrogen peroxide (H2O2) damaged B. mori cells, which were separated from the ovary of B. mori. In addition, comparisons were made between damage caused by H2O2 and by ultraviolet (UV) irradiation. The results showed that the potential change of the mitochondrial membrane occurred at 0.5 h after H2O2 stimulation, which was sooner than occurred in the UV treated model where the obvious decrease appeared at 6 h after stimulation. In addition, the total change in the potential of the mitochondrial membrane in H2O2 treated B. mori cells was larger than with UV treated cells during the whole process. Analysis of fluorescent quantitative real-time PCR demonstrated that ice-2 and ice-5 might be involved in both H2O2 and UV-induced apoptosis in B. mori cells. Notably, after exposure to H2O2, the expression patterns of ice-5 were remarkably higher than those of ice-2, while the result was the opposite after exposure to UV irradiation. The data indicate that apoptosis induced by H2O2 was directly related to the mitochondrial pathway. The two isoforms of B. mori ice may play different roles in the mitochondrion associated apoptotic pathway in B. mori cells, and the apoptotic pathway in H2O2 induced B. mori cells is different from the UV induced apoptotic pathway.

Citation

PMID: 20572791 [PubMed - in process]

 

Title

Transcriptional regulation of the gene for prothoracico tropic hormone in the silkworm, Bombyx mori.

Authors

Wei ZJ, Yu M, Tang SM, Yi YZ, Hong GY, Jiang ST.

 

Department of Biotechnology, Hefei University of Technology, Hefei, 230009, People's Republic of China, zjwei@hfut.edu.cn.

Journal

Mol Biol Rep. 2010 Jun 19. [Epub ahead of print]

Abstract

Prothoracicotropic hormone (PTTH) is one of key players in regulation of insect growth, molting, metamorphosis, diapause, and is expressed specifically in the two pairs of lateral PTTH-producing neurosecretory cells in the brain. Analysis of cis-regulatory elements of the PTTH promoter might elucidate the regulatory mechanism controlling PTTH expression. In this study, the PTTH gene promoter of Bombyx mori (Bom-PTTH) was cloned and sequenced. The cis-regulatory elements in Bom-PTTH gene promoter were predicted using Matinspector software, including myocyte-specific enhancer factor 2, pre-B-cell leukemia homeobox 1, TATA box, etc. Transient transfection assays using a series of fragments linked to the luciferase reporter gene indicated that the fragment spanning -110 to +33 bp of the Bom-PTTH promoter showed high ability to support reporter gene expression, but the region of +34 to +192 bp and -512 to -111 bp repressed the promoter activity in the BmN and Bm5 cell lines. Electrophoretic mobility shift assays demonstrated that the nuclear protein could specifically bind to the region spanning -124 to -6 bp of the Bom-PTTH promoter. Furthermore, we observed that the nuclear protein could specifically bind to the -59 to -30 bp region of the Bom-PTTH promoter. A classical TATA box, TATATAA, localized at positions -47 to -41 bp, which is a potential site for interaction with TATA box binding protein (TBP). Mutation of this TATA box resulted in no distinct binding band. Taken together, TATA box was involved in regulation of PTTH gene expression in B. mori.

Citation

PMID: 20563654 [PubMed - as supplied by publisher]

 

Title

Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides.

Authors

Sakamoto S, Pongkitwitoon B, Nakamura S, Maenaka K, Tanaka H, Morimoto S.

 

Department of Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

Journal

J Biochem. 2010 Jun 30. [Epub ahead of print]

Abstract

A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence (HMSS) to accelerate secretion of the recombinant GRe-scFv into the hemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the hemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg per liter of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming refolding when it expressed in Escherichia coli.

Citation

PMID: 20592135 [PubMed - as supplied by publisher]

 

Title

Cloning and characterization of a beta-N-acetylglucosaminidase (Bm FDL) from silkworm Bombyx mori.

Authors

Nomura T, Ikeda M, Ishiyama S, Mita K, Tamura T, Okada T, Fujiyama K, Usami A.

 

Research Institute of Biological Science, Katakura industries CO., LTD., 1548 Simo-okutomi, Sayama, Saitama 350-1332, Japan.

Journal

J Biosci Bioeng. 2010 May 26. [Epub ahead of print]

Abstract

In insects, beta-N-acetylglucosaminidase (GlcNAcase) participates in critical physiological processes such as fertilization, metamorphosis, and glycoconjugate degradation. Insects produce glycoproteins carrying paucimannosidic-type N-glycans, the terminal GlcNAc residue of which is cleaved by a GlcNAc-linkage specific GlcNAcase, also known as the fused lobes (FDL) protein. To obtain information on the structure of GlcNAcases and insight into their contribution to physiological processes, we cloned Bombyx mori FDL (BmFDL) from silkworm larvae. The full-length cDNA (1.9kb) encoded a protein of 633 amino acids with 42% amino acid sequence identity to Drosophila melanogaster FDL (DmFDL). Recombinant BmFDL cleaved only beta-1,2-linked GlcNAc residues from the alpha-1,3 branch of biantennary N-glycan. This substrate specificity was similar to that of DmFDL. Microsomal FDL activity was inhibited by anti-BmFDL antibodies. Taken together, our results suggest that BmFDL is a N-glycan-processing GlcNAcase in B. mori. Copyright © 2010 Elsevier B.V. All rights reserved.

Citation

PMID: 20547376 [PubMed - as supplied by publisher]

 

Title

Genomic analysis of carboxyl/cholinesterase genes in the silkworm Bombyx mor i. Free Article

Authors

Tsubota T, Shiotsuki T.

Journal

BMC Genomics. 2010 Jun 14;11(1):377. [Epub ahead of print]

Abstract

ABSTRACT: BACKGROUND: Carboxyl/cholinesterases (CCEs) have pivotal roles in dietary detoxification, pheromone or hormone degradation and neurodevelopment. The recent completion of genome projects in various insect species has led to the identification of multiple CCEs with unknown functions. Here, we analyzed the phylogeny, expression and genomic distribution of 69 putative CCEs in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). RESULTS: A phylogenetic tree of CCEs in B. mori and other lepidopteran species was constructed. The expression pattern of each B. mori CCE was also investigated by a search of an expressed sequence tag (EST) database, and the relationship between phylogeny and expression was analyzed. A large number of B. mori CCEs were identified from a midgut EST library. CCEs expressed in the midgut formed a cluster in the phylogenetic tree that included not only B. mori genes but also those of other lepidopteran species. The silkworm, and possibly also other lepidopteran species, has a large number of CCEs, and this might be a consequence of the large cluster of midgut CCEs. Investigation of intron-exon organization in B. mori CCEs revealed that their positions and splicing site phases were strongly conserved. Several B. mori CCEs, including juvenile hormone esterase, not only showed clustering in the phylogenetic tree but were also closely located on silkworm chromosomes. We investigated the phylogeny and microsynteny of neuroligins in detail, among many CCEs. Interestingly, we found the evolution of this gene appeared not to be conserved between B. mori and other insect orders. CONCLUSIONS: We analyzed 69 putative CCEs from B. mori. Comparison of these CCEs with other lepidopteran CCEs indicated that they had conserved expression and function in this insect order. The analyses showed that CCEs were unevenly distributed across the genome of B. mori and suggested that neuroligins may have a distinct evolutionary history from other insect order. It is possible that such an uneven genomic distribution and a unique neuroligin evolution are shared with other lepidopteran insects. Our genomic analysis has provided novel information on the CCEs of the silkworm, which will be of value to understanding the biology, physiology and evolution of insect CCEs.

Citation

PMID: 20546589 [PubMed - as supplied by publisher]

 

Title

Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument of the silkworm, Bombyx mori.

Authors

Fujii T, Daimon T, Uchino K, Banno Y, Katsuma S, Sezutsu H, Tamura T, Shimada T.

 

Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

Journal

Insect Mol Biol. 2010 Jun 7. [Epub ahead of print]

Abstract

Abstract The larval integument of the silkworm, Bombyx mori, is opaque because urate granules accumulate in the epidermis. Although the biosynthetic pathway of uric acid is well studied, little is known about how uric acid accumulates as urate granules in epidermal cells. In the distinct oily (od) mutant silkworm, the larval integument is translucent because of the inability to construct urate granules. Recently, we have found that the od mutant has a genomic deletion in the B. mori homologue of the human biogenesis of lysosome-related organelles complex1, subunit 2 (BLOS2) gene (BmBLOS2). Here, we performed a molecular and functional characterization of BmBLOS2. Northern blot analysis showed that BmBLOS2 was ubiquitously expressed in various tissues. We analysed the structure of a newly isolated mutant (od(B)) allelic to od and found a premature stop codon in the coding sequence of BmBLOS2 in this new mutation. Moreover, the translucent phenotype was rescued by the germ-line transformation of the wild-type BmBLOS2 allele into the od mutant. Our results suggest that BmBLOS2 is responsible for the od mutant phenotype and plays a crucial role in biogenesis of urate granules in the larval epidermis of the silkworm. The relationships amongst Hermansky-Pudlak syndrome (HPS) genes in mammals, granule group genes in Drosophila and translucent mutant genes in B. mori are discussed.

Citation

PMID: 20546041 [PubMed - as supplied by publisher]

 

Title

The relationship between internal domain sequences of piggyBac and its transposition efficiency in BmN cells and Bombyx mori. Free Article

Authors

Zhuang L, Wei H, Lu C, Zhong B.

 

Zhejiang University, Hangzhou, China.

Journal

Acta Biochim Biophys Sin (Shanghai). 2010 Jun 15;42(6):426-31.

Abstract

The piggyBac transposon, which includes terminal inverted repeat sequences and internal domain (ID) sequences, is widely used as a tool for insect transformation. To optimize this system for transgenic research on Bombyx mori, we examined the effects of the amount of the transposase plasmid and its ID sequences on the expression of green fluorescent protein (GFP). Four kinds of transposon plasmids, pB[A3GFP]-1 with the full length of ID sequences, pB[A3GFP]-2 having only the 3' ID sequence, pB[A3GFP]-3 without ID sequences, and pB[A3GFP]-4 containing 333 bp of the 5' ID sequence, and 179 bp of the 3' ID sequence were constructed with GFP as the marker. After transfecting these four plasmids into BmN cells, we analyzed the transfecting efficiency by comparing the GFP positive to negative cell ratio. Our results indicated that plasmid pB-4 got the highest ratio on the 22nd day. Moreover, the GFP positive to negative cell ratio increased with higher amount of transposase plasmid without overproduction inhibition. Furthermore, we injected three piggyBac transposon plasmids, pB[A3GFP]-1, pB[3xP3GFP]-3, and pB[3xP3RFP]-4 harboring different markers into preblastoderm stage eggs of B. mori, and found that the transformation efficiency of pB[3xP3RFP]-4 was 3.8 folds higher than pB[A3GFP]-1, whereas pB[3xP3GFP]-3 failed to produce transformants. Our results suggested that pB-4 may be one of the best piggyBac transposon plasmids currently available for germline transformation in B. mori.

Citation

PMID: 20539943 [PubMed - in process]

 

Title

The silkworm Green b locus encodes a quercet in 5-O-glucosyltransferase that produces green cocoons with UV-shielding properties.

Authors

Daimon T, Hirayama C, Kanai M, Ruike Y, Meng Y, Kosegawa E, Nakamura M, Tsujimoto G, Katsuma S, Shimada T.

 

Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.

Journal

Proc Natl Acad Sci U S A. 2010 Jun 22;107(25):11471-6. Epub 2010 Jun 4.

Abstract

In the silkworm Bombyx mori, dietary flavonoids are metabolized and accumulate in cocoons, thereby causing green coloration. Classical genetic studies suggest that more than seven independent loci are associated with this trait; however, because of the complex inheritance pattern, none of these loci have been characterized molecularly, and a plausible and comprehensive model for their action has not been proposed. Here, we report the identification of the gene responsible for the Green b (Gb) locus involving the green cocoon trait. In +(Gb) animals, glucosylation at the 5-O position of dietary quercetin did not occur, and the total amount of flavonoids in tissues and cocoons was dramatically reduced. We performed positional cloning of Gb and found a 38-kb deletion in a UDP-glucosyltransferase (UGT) gene cluster associated with the +(Gb) allele. RT-PCR and biochemical studies suggested that deletion of Bm-UGT10286 (UGT) is responsible for Gb and Bm-UGT10286 is virtually the sole source of UGT activity toward the 5-O position of quercetin. Our data show that the regiospecific glucosylation of flavonoids by the quercetin 5-O-glucosyltransferase can greatly affect the overall bioavailability of flavonoids in animals. Furthermore, we provide evidence that flavonoids increase the UV-shielding activity of cocoons and thus could confer an increased survival advantage to insects contained in these cocoons. This study will lead to greater understanding of mechanisms for metabolism, uptake, and transport of dietary flavonoids, which have a variety of biological activities in animals and beneficial effects on human health.

Citation

PMID: 20534444 [PubMed - in process]

 

Title

Characterization of a germination-accelerating factor from the silkworm ( Bombyx mori Linnaeus) of entomopathogenic fungus Nomuraea rileyi (Farlow) Samson. Free Article

Authors

Noda T, Ono M, Iimure K, Araki T.

 

Kumamoto Prefectural Agriculture Research Center, Koshi, Kumamoto, Japan. noda-t-dw@pref.kumamoto.lg.jp

Journal

Biosci Biotechnol Biochem. 2010 Jun 23;74(6):1226-30. Epub 2010 Jun 7.

Abstract

The conidium of the entomopathogenic fungus, Nomuraea rileyi, has been found to germinate rapidly in the presence of a host insect-derived extract. This extract therefore appears to contain an important factor involved in host recognition by N. rileyi, although the substance (germination-accelerating factor, GAF) responsible for such unique germination behavior has yet to be identified. Our previous study was extended to the isolation of GAF from pupae of the silkworm, a host insect of N. rileyi. This present work subjects GAF to a structural analysis. The chemical structure of GAF is characterized as 2S-amino-tetradeca-4-ene-1,3R-diol (D-erythro-C(14)-sphingosine) based on spectroscopic data. An examination of the structure-activity relationship shows that the activity of D-erythro-C(14)-sphingosine was superior to that of sphingosines with shorter and longer carbon chains. It is suggested that the molecular species with a 14-carbon chain of a sphingosine is important for host recognition.

Citation

PMID: 20530914 [PubMed - in process]

 

Title

Production of a non-triple helical collagen alpha chain in transgenic silkworms and its evaluation as a gelatin substitute for cell culture.

Authors

Adachi T, Wang X, Murata T, Obara M, Akutsu H, Machida M, Umezawa A, Tomita M.

 

Neosilk Co., Ltd., 3-13-26 Kagamiyama, Higashihiroshima, Hiroshima 739-0046, Japan; telephone: +81 82 431 0652; fax: +81 82 431 0654.

Journal

Biotechnol Bioeng. 2010 Apr 8;106(6):860-870. [Epub ahead of print]

Abstract

We generated transgenic silkworms that synthesized human type I collagen alpha1 chain [alpha1(I) chain] in the middle silk glands and secreted it into cocoons. The initial content of the recombinant alpha1(I) chain in the cocoons of the transgenic silkworms was 0.8%. The IE1 gene, a trans-activator from the baculovirus, was introduced into the transgenic silkworm to increase the content of the chain. We also generated silkworms homozygous for the transgenes. These manipulations increased the alpha1(I) chain content to 8.0% (4.24 mg per cocoon). The alpha1(I) chain was extracted and purified from the cocoons using a very simple method. The alpha1(I) chain contained no hydroxyprolines due to the absence of prolyl-hydroxylase activity in the silk glands. Circular dichroism analysis showed that the secondary structure of the alpha1(I) chain is similar to that of denatured type I collagen, demonstrating the absence of the triple helical structure. Human skin fibroblasts were seeded on the alpha1(I) chain-coated dishes. The cells attached and spread, although at decreased chain concentrations the spreading rate was lower than that of the collagen and gelatin. Cynomolgus monkey embryonic stem cells cultured on the alpha1(I) chain-coated dishes maintained an undifferentiated state after 30 passages, and their pluripotency was confirmed by teratoma formation in severe combined immunodeficient mice. These results show that the recombinant human alpha1(I) chain is a promising candidate biomaterial as a high-quality and safe gelatin substitute for cell culture. Biotechnol. Bioeng. 2010;106: 860-870. (c) 2010 Wiley Periodicals, Inc.

Citation

PMID: 20589667 [PubMed - as supplied by publisher]

 

Title

Quantitative cell-based reporter gene assays using droplet-based microfluidics.

Authors

Baret JC, Beck Y, Billas-Massobrio I, Moras D, Griffiths AD.

 

Institut de Science et d'Ingénierie Supramoléculaires, Université de Strasbourg, CNRS UMR 7006, 8 Allée Gaspard Monge, BP 70028, F-67083 Strasbourg Cedex, France. jc.baret@unistra.fr

Journal

Chem Biol. 2010 May 28;17(5):528-36.

Abstract

We used a droplet-based microfluidic system to perform a quantitative cell-based reporter gene assay for a nuclear receptor ligand. Single Bombyx mori cells are compartmentalized in nanoliter droplets which function as microreactors with a >1000-fold smaller volume than a microtiter-plate well, together with eight or ten discrete concentrations of 20-hydroxyecdysone, generated by on-chip dilution over 3 decades and encoded by a fluorescent label. The simultaneous measurement of the expression of green fluorescent protein by the reporter gene and of the fluorescent label allows construction of the dose-response profile of the hormone at the single-cell level. Screening approximately 7500 cells per concentration provides statistically relevant data that allow precise measurement of the EC(50) (70 nM +/- 12%, alpha = 0.05), in agreement with standard methods as well as with literature data. 2010 Elsevier Ltd. All rights reserved.

Citation

PMID: 20534350 [PubMed - in process]

 

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