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Expression of Human Stem Cell Factor with Recombinant Baculovirus in BmN Cell Line and Silkworm
Xijie, Guo,Yongfeng, Jin,Mingguan, Yang,Yaozhou, Zhang Korean Society of Sericultural Science 2002 International Journal of Industrial Entomology Vol.4 No.1
A recombinant transfer vector pBacSCF was constructed by inserting huamn stem cell factor (hSCF) cDNA into plasmid pBacPAK8. BmN cells were co-transfected with modified Bombyx mori, nuclear polyhedrosis virus (BmBacPAK) DNA and the recmbinant transfer vector to construct a recombinant baculovirus containing hSCE gene. DNA dot blotting and RNA dot blotting demonstrated that the hSCE gene was contained in the recombinant virus and transcribed. The recombinant baculovirus was infectious to BmN cells and to silkworm. SDS-PAGE analysis showed a specific band of expressed product in the extract of infected cells and in the heamolymph of infected larvae. Bioactivity of the recombinant hSCE was determined with W-1 cell line and MTT colorimetric method in synergy with interlukin-3 (IL-3). These results revealed that the hSCF gene was over-expressed in cultured cells and lavae of silkworm.
Molecular Cloning of a Profilin cDNA from Bombyx mori
Wei, Yadong,Gui, Zhongzheng,Choi, Young Soo,Guo, Xijie,Zhang, Guozheng,Sohn, Hung Dae,Jin, Byung Rae Korean Society of Sericultural Science 2004 International Journal of Industrial Entomology Vol.9 No.1
The actin-binding protein profilin cDNA was firstly isolated from the lepidopteran insect, silkworm Bombyx mori. The B. mori profilin cDNA contains an open reading frame of 378 bp encoding 126 amino acid residues and possesses three cysteine residues. The deduced amino acid sequence of the B. mori profilin cDNA showed 80% identity to Apis mellifera profilin and 72% to Drosophila melanogaster profilin. Northern blot analysis showed that B. mori profilin is highly expressed in epidermis and less strongly in silk gland. In addition, Northern blot analysis revealed the presence of B. mori profilin transcripts in all tissues examined, suggesting that B. mori profilin gene is expressed in most, if not all, body tissues.
Tao Geng,Yuxia Huang,Chengxiang Hou,Guangxing Qin,Dingding Lv,Xijie Guo 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.3
Innate immunity is critical to insects and plays an important role in pathogen elimination and wound healing. Toll signaling pathway is the major signaling pathway associated with insect innate immunity mediating synthesis of anti-fungal/bacterial peptides. To better understand the Toll signaling pathwaymediated immune response in Bombyx mori against Beauveria bassiana infection, expression patterns of genes encoding sixteen putative components of Toll signaling pathway in the silkworm larvae challenged with B. bassiana, including four pattern recognition receptors (PRRs, i.e. BmβGRP 1, 2, 3, 4), eight Toll-like receptors (TLRs, i.e. Bm18w, BmToll 1, 3, 6, 9, 7, 10, 11) and four effectors (BmMoricin 1, BmGloverin 2, BmDefensin 1 and BmLysozyme 1), were analyzed using quantitative real-time PCR. At the same time, the changes in their expression by RNAi knock-down of the four PRRswere also detected.Moreover, the effects of Toll signaling pathway inhibitors on antifungal activity in larvae hemolymphwere also analyzed. The results showed that the expression levels of genes encoding sixteen putative components of Toll signaling pathway were obviously altered by the challengewith B. bassiana, but their temporal regulation mode was significantly different. Based on the expression patterns of the genes related to Toll signaling pathway, two sub-paths of immune signal recognition and transduction might be proposed in the response of silkworm larvae against B. bassiana infection. Besides, Toll signaling pathway inhibitor could significantly inhibit the antifungal activity in hemolymph and resulted in increased sensitivity of silkworm larvae to the B. bassiana infection, while the treatment with heat-inactivated B. bassiana could induce antifungal activity in the hemolymph and led to stronger resistance of the silkworm. These results implied that Toll signaling pathway played important roles in the antifungal immune response of the silkworm larvae, in which different components of Toll signaling pathway might play a specific regulatory function. These findings yield insights into the innate immune mechanisms underlying Toll signaling pathway in silkworm.
Characterization and profiling of MicroRNAs in posterior silk gland of the silkworm (Bombyx mori)
Fei Song,Xin Wang,Chen Chen,Yangyang Fan,Shunming Tang,Jinshan Huang,Xijie Guo,Xingjia Shen 한국유전학회 2015 Genes & Genomics Vol.37 No.8
MicroRNAs (miRNAs) regulate expression of genes at post-transcriptional level by binding on complementary sequences of target mRNAs and play multiple roles in biological processes. To investigate the differential expression of miRNAs in posterior silk gland (PSG) of silkworm (Bombyx mori) in different periods and regulation of miRNAs on the expression of fibroin genes, Solexa sequencing technology was used to detect miRNAs in PSGs of fourth-instar day-2 larvae and fifth-instar day-3 larvae, respectively. As a result, 466 previously reported miRNAs, and 35 novel miRNAs were detected, and 499 of these detected miRNAs are predicted to target 13,383 genes by target prediction softwares. Additionally, 29 miRNAs expressed differently between the PSG of fourthinstar day-2 larvae and fifth-instar day-3 larvae were found, and the differential expression of these miRNAs may play an important role in the expression of fibroin genes.
Zheng Gui Zhong,Kim Bo-Yeon,Yoon Hyung-Joo,Wei Ya Dong,Xijie Guo,Jin Byung-Rae,Shon Hung-Dae Korean Society of Sericultural Science 2007 International Journal of Industrial Entomology Vol.14 No.1
In a previous study, three larval cuticle protein genes were cloned from the mulberry longicorn beetle, Apriona germari (Comp. Biochem. Physiol. B 136, 803-811, 2003). In the present study, the genomic structures of these three larval cuticle protein genes (AgLCP9.2, AgLCP12.6 and AgLCP12.3) were elucidated. All three cuticle protein genes consist of one intron and two exons. Southern blot analysis of genomic DNA suggested that three cuticle protein genes are a single copy gene. In addition, a novel larval cuticle protein gene, AgLCP10.6, was cloned from A. germari in this study. The AgLCP10.6 cDNA contains an ORF of 300 nucleotides that are capable of encoding a 100-amino acid polypeptide with a predicted molecular mass of 10.6 kDa. The amino acid sequence deduced from the AgLCP10.6 cDNA contained a type-specific consensus sequence identifiable in other insect cuticle proteins and is most homologous to Drosophila melanogaster cuticle protein ACP65A (51 % protein sequence identity). Northern blot analysis revealed that AgLCP10.6 showed epidermis-specific expression.
Zhao, Yuan,Chen, Kepin,Yao, Qing,Wu, Yang-Chun,Zhang, Jian,Guo, Xijie Korean Society of Sericultural Science 2006 International Journal of Industrial Entomology Vol.13 No.2
Seveval Chinese and Japanese varieties with good characters were used in the breeding. After 5 years (15 generations), a pair of robust and high quality silkworm variety with NPV resistance was bred by means of a combination of crossing and pedigree selection complemented by the selection of NPV resistance. The variety was identified jointly nationwide in 2003 and 2004, and appraised by National Mulberry and Silkworm Appraising Committee. Results are as follows: its cocooning rate is over 93%, shell rate 23-25%, filament length 1200-1300 meters, reelability 75-88%, Length of non-broken cocoon filament 900-1100 meters, raw silk rate 17-19%, neatness 95-97 points, and cocoon crop, cocoon shell weight and raw silk weight per 10000 larvae is higher than those of the control variety by 7-10%, 14-19% and 14-18%, respectively. The variety is not only robust, resistant to high temperature and NPV, easy to rear, uniform in hatching, molting and maturing, but also lays more eggs, and its fecundity is high. It is suitable to rear in the Yangtze River Basin, the Yellow River basin and the Pearl River basin of China.
Feng Ming Zou,이광식,김보연,김홍자,Zhong Zheng Gui,Guozheng Zhang,Xijie Guo,진병래 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.4
Insect cuticular melanization is regulated by the prophenoloxidase (proPO)-activating system, which is also involved in the innate immune reaction. Here, we demonstrate how the differentiation of the proPO-activating system is regulated toward a cuticular melanization or innate immunity function in silkworm (Bombyx mori) pupae. Our results indicate that the differential and spatial regulation of key components, such as the proPOactivating enzyme and proPOs, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism that is mediated by the priming of the proPO system.
Kun Gao,Xiang Yuan Deng,Meng Ke Shang,Heying Qian,Xijie Guo 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.3
The cDNA of a biphenyl hydrolase-like (BPHL) protein from Bombyx mori was cloned via rapid amplification of cDNA ends and submitted to GenBank under accession number JN020647. The full-length BmBPHL cDNA was 1161 bp, with four exons and three introns. It consisted of a 208 bp 5′-terminal untranslated region (UTR) and a 191 bp 3′-UTR with three polyadenylation signal sequences AATAAA and a poly(A) tail. The BmBPHL cDNA encodes a 253–amino acid polypeptide with a theoretical isoelectric point of 8.67 and a predicted molecular weight of 28.9 kDa. The deduced amino acid sequence of BmBPHL contained an abhydrolase_6 domain and the Gly-X-Ser-X-Gly motif that is characteristic of serine hydrolases. Sequence comparison showed that BmBPHL is 51% identical to Tribolium castaneum BPHL and 50% identical to Nasonia vitripennis BPHL. Phylogenetic analysis revealed that BmBPHL is grouped with insect BPHL proteins, separating from vertebrate BPHLs. The BmBPHL mRNA transcripts were mainly detected in hemolymph and fat body using fluorescent quantitative real-time PCR. In addition, infectionwith B. mori cytoplasmic polyhedrosis virus (BmCPV) upregulated the relative BmBPHL expression in the hemolymph and midgut. Therefore, BmBPHL may have an important function in the response of silkworms to BmCPV infection.
Molecular Cloning of a LIM Protein cDNA from the Mulberry Longicorn Beetle, Apriona germari
Gui, Zhongzheng,Wei, Yadong,Yoon, Hyung Joo,Kim, Iksoo,Guo, Xijie,Jin, Byung Rae,Sohn, Hung Dae Korean Society of Sericultural Science 2004 International Journal of Industrial Entomology Vol.9 No.1
Here we report the molecular cloning of a LIM protein cDNA of the CRP (cysteine-rich protein) family from the mulberry longicorn beetle, Apriona, geramri. The A. germari LIM protein cDNA contains an open reading frame of 276 bp encoding 92 amino acid residues with a calculated molecular weight of approximately 10 kDa. The A. germari LIM protein contains the cysteine-rich consensus sequence of LIM domain and the glycine-rich consensus sequence observed in cysteine-rich protein family 1 (CRP1). The potential nuclear targeting signal is retained. The deduced amino acid sequence of the A. germari LIM protein cDNA showed 81 % identity to both Bombyx mori muscle LIM protein (Mlp) and Drosophila melanogaster Mlp60A and 77% to Epiblema scudderiana Mlp. Northern blot analysis showed that A. germari LIM protein is highly expressed in epidermis and muscle, and less strongly in midgut, but not in the fat body.
PCR-Based Detection of Densovirus Infection in Silkworm (Bombyx mori L.)
Hou Chengxiang,Li Muwang,Gui Zhongzheng,Xu Anying,Guo Xijie Korean Society of Sericultural Science 2005 International Journal of Industrial Entomology Vol.11 No.2
Two pairs of DNA primers were designed for the detection of the Zhenjiang (China) strain of Bombyx mori densonucleosis virus (BmDNV-Z). These primers were designed from the nucleotide sequence of major structural protein gene (putative VD1-ORF2). PCR amplification was attempted from different issues (including silk gland, blood, skin and midgut) and feces of the silkworm which infected wit BmDNV-Z were amplified by PCR. Both of the primers gave expected size of in the DNA bands from midgut and feces, but not in the DNA of silk gland, blood and skin. The two bands were sequenced, and their sequence were same as the sequence designed for. BmDNV-Z could be successfully detected in single silkworm after it was infected for 12 hrs, and could not be detected before 9 hrs after infected.