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检索条件"任意字段=中国蚕学会第十二届家柞蚕遗传育种与良种繁育学术研讨会"

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Inhibition of BmNPV replication in silkworm cells using inducible and regulated artificial microRNA precursors targeting the essential viral gene lef-11

Inhibition of BmNPV replication in silkworm cells using indu...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Jun Zhang Qian He Chun-Dong Zhang Xiang-Yun Chen Xue-Mei Chen Zhan-Qi Dong Na Li Xiu-Xiu Kuang Ming-Ya Cao Cheng Lu Min-Hui Pan State Key Laboratory of Silkworm Genome Biology Southwest University Department of Biochemistry and Molecular Biology Chongqing Medical University Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry Southwest University

Bombyx mori nucleopolyhedrovirus(Bm NPV) is a major silkworm pathogen, causing substantial economic losses to the sericulture industry annually. We demonstrate a novel anti-Bm NPV system expressing mature artificial micro RNAs(ami RNAs) targeting the viral lef-11 gene. The mature amiRNAs inhibited the lef-11 gene in silkworm BmN-SWU1 cells. Antiviral assays demonstrated that mature ami RNAs silenced the gene and inhibited BmNPV proliferation efficiently. As constitutive overexpression of mature ami RNAs may induce acute cellular toxicity, we further developed a novel virus-induced ami RNA expression system. The amiRNA cassette is regulated by a baculovirus-induced fusion promoter. This baculovirus-induced RNA interference system is strictly regulated by virus infection, which functions in a negative feedback loop to activate the expression of mature amiRNAs against lef-11 and subsequently control inhibition of BmNPV replication. Our study advances the use of a regulatable amiRNA cassette as a safe and effective tool for research of basic insect biology and antiviral application.

关键词： BmNPV RNA interference Artificial microRNA Baculovirus-induced RNA interference system

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A composite method for mapping quantitative trait loci without interference of female achiasmatic and gender effects in silkworm, Bombyx mori

A composite method for mapping quantitative trait loci witho...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： C.Li W.Zuo X.Tong H.Hu L.Qiao J.Song G.Xiong R.Gao F.Dai C.Lu State Key Laboratory of Silkworm Genome Biology Southwest University Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry Southwest University Institute of Entomology and Molecular Biology College of Life Sciences Chongqing Normal University

The silkworm, Bombyx mori, is an economically important insect that was domesticated more than 5000 years ago. Its major economic traits focused on by breeders are quantitative traits, and an accurate and efficient QTL mapping method is necessary to explore their genetic architecture. However, current widely used QTL mapping models are not well suited for silkworm because they ignore female achiasmate and gender effects. In this study, we propose a composite method combining rational population selection and special mapping methods to map QTL in silkworm. By determining QTL for cocoon shell weight(CSW), we demonstrated the effectiveness of this method. In the CSW mapping process, only 56 markers were used and five loci or chromosomes were detected, more than in previous studies. Additionally, loci on chromosomes 1 and 11 dominated and accounted for 35.10% and 15.03% of the phenotypic variance respectively. Unlike previous studies, epistasis was detected between loci on chromosomes 11 and 22. These mapping results demonstrate the power and convenience of this method for QTL mapping in silkworm, and this method may inspire the development of similar approaches for other species with special genetic characteristics.

关键词： Bombyx mori cocoon shell weight genetic architecture population selection quantitative trait loci mapping

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Expression Pattern of a Trypsin-like Serine Protease Gene from the Chinese oak silkworm, Antheraea pernyi upon Pathogen Invasion

Expression Pattern of a Trypsin-like Serine Protease Gene fr...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者：王勇 Liu Wei Jiang Yiren Li Donghua 秦利 College of Bioscience and Biotechnology Shenyang Agricultural University Liaoning Engineering and Technology Resource Center for Insect Resource

Trypsin-like serine protease(TLS) was a member of the chymotrypsin/trypsin superfamily enzymes. In order to research the function of TLS in Chinese oak silkworm Antheraea pernyi, TLS gene was cloned in this study and named as ApTLS(Gen Bank accession No. KF779933). A 1020 bp full-length cDNA sequence of ApTLS including an open reading frame(ORF) of 774 bp coding for a theoretical 257 amino acids protein. Bioinformatic analysis revealed that the gene with a 20 residues N-terminal signal peptide, six conservative cysteine residues and a conserved C-terminal domain, which belongs to the serine protease superfamily. Semi-quantitative RT-PCR indicated that ApTLS was widely distributed in all life-history stages, and the midgut has the highest expression level in A. pernyi. Quantitative Real time PCR(qRT-PCR) was employed to further identify the expression patterns of ApTLS gene in the midgut of A. pernyi after different exogenous microorganisms infected. Nuclear polyhedrosis virus(Ap NPV), Nosema pernyi, Streptococcus pernyi and Beauveria bassiana were used as the exogenous microorganisms in order to test the ApTLS gene expression. All of them can induce the gene up-regulated compared to the control group. Through the expression pattern of the ApTLS gene, we hope this results can led a better understanding about innate immune system of A. pernyi.

关键词： Antheraea pernyi trypsin-like serine protease Insect immunity Gene expression

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DNA Synthesis during Endomitosis Is Stimulated by Insulin via the PI3K/Akt and TOR Signaling Pathways in the Silk Gland Cells of Bombyx mori

DNA Synthesis during Endomitosis Is Stimulated by Insulin vi...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Yaofeng Li Xiangyun Chen Xiaofang Tang Chundong Zhang La Wang Peng Chen Minhui Pan Cheng Lu State Key Laboratory of Silkworm Genome Biology Southwest University Department of Biochemistry and Molecular Biology Chongqing Medical University Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry Southwest University

Silk gland cells undergo multiple endomitotic cell cycles during silkworm larval ontogeny. Our previous study demonstrated that feeding is required for continued endomitosis in the silk gland cells of silkworm larvae. Furthermore, the insulin signaling pathway is closely related to nutritional signals. To investigate whether the insulin signaling pathway is involved in endomitosis in silk gland cells, in this study, we initially analyzed the effects of bovine insulin on DNA synthesis in endomitotic silk gland cells using 5-bromo-2'-deoxyuridine(Brd U) labeling technology, and found that bovine insulin can stimulate DNA synthesis. Insulin signal transduction is mainly mediated via phosphoinositide 3-kinase(PI3K)/Akt, the target of rapamycin(TOR) and the extracellular signal-regulated kinase(ERK) pathways in vertebrates. We ascertained that these three pathways are involved in DNA synthesis in endomitotic silk gland cells using specific inhibitors against each pathway. Moreover, we investigated whether these three pathways are involved in insulin-stimulated DNA synthesis in endomitotic silk gland cells, and found that the PI3K/Akt and TOR pathways, but not the ERK pathway, are involved in this process. These results provide an important theoretical foundation for the further investigations of the mechanism underlying efficient endomitosis in silk gland cells.

关键词： Bombyx mori silk gland DNA synthesis insulin signaling

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Bombyx mori nucleopolyhedrovirus ORF79 is a per os infectivity factor associated with the PIF complex

Bombyx mori nucleopolyhedrovirus ORF79 is a per os infectivi...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Zhan-Qi Dong Jun zhang Xue-Mei Chen Qian He Ming-Ya Cao La Wang Hai-Qing Li Wen-Fu Xiao Cai-Xia Pan Cheng Lu Min-Hui Pan State Key Laboratory of Silkworm Genome Biology Southwest University Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry Southwest University

Bombyx mori nucleopolyhedrovirus(BmNPV) ORF79(Bm79) encodes an occlusion-derived virus(ODV)-specific envelope protein, which is a homologue of the per os infectivity factor 4(PIF4) of Autographa californica multiple nucleopolyhedrovirus(Ac MNPV). To investigate the role of ORF79 in the BmNPV life cycle, a Bm79 knockout virus(vBmBm79KO) was constructed through homologous recombination in Escherichia coli. Viral DNA replication, budded virus(BV) production and polyhedral formation were unaffected by the absence of BM79. However, results of the larval bioassay demonstrated that the Bm79 deletion resulted in a complete loss of per os infection. Immunofluorescence analysis showed that BM79 localized at the innernuclear membrane of infected cells through its N-terminal sorting motif(SM). Further bimolecular fluorescence protein complementation and co-immunoprecipitation assays demonstrated the interaction of BM79 with PIF1, PIF2, PIF3 and ODV-E66. Thus, BM79 plays an important role in per os infection and is associated with the viral PIF complex of Bm NPV.

关键词： Bombyx mori BmNPV Bm79 Per os infection PIF complex

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Intestinal microecology associated with fluoride resistance capability of the silkworm（Bombyx mori L.）

Intestinal microecology associated with fluoride resistance ...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Guan-Nan Li Xue-Juan Xia Wen-Chao Tang Yong Zhu College of Biotechnology Southwest University

The silkworm（Bombyx mori L.） is an ideal model of Lepidoptera. However, the diversity and function of the intestinal microbiota in the gut of silkworm remain largely unknown. Changes in the intestinal microecology in fluoride-resistant strain T6 and fluoride-susceptible strain 734 of the silkworm in response to fluoride exposure were investigated. T6 and 734 were treated with 200 mg/kg fluoride（designated as T6-T and 734-T groups） and deionized water（designated as T6-C and 734-C groups）. Culture-dependent approach revealed that the numbers of intestinal bacteria in the 734-T group significantly decreased compared with that in the 734-C group（4.8 ± 0.6 × 107 CFU/m L vs. 7.5± 0.7 × 107 CFU/m L;P ＜ 0.05）. Analyses of the intestinal con-tent p H showed that the p H decreased in the 734-T group only. Additionally, SCFA concentrations significantly decreased in both treatment groups compared with the control groups. High-throughput sequencing indicated that the intestinal mi-crobiota in the 734-T group was significantly more diverse than those in the other groups. The bacterial community was composed of two dominant groups（Firmicutes and Proteobacteria）. Principal component analyses revealed a sig-nificant difference in the composition of the intestinal micro-biota in the 734-T group compared with those in the other groups. Thaumarchaeota and Euryarchaeota were more abundant in the 734-T group, but they were less abundant in the other groups. This study enhances our understanding about the diversity and function of silkworm intestinal micro-biota in response to fluoride exposure among silkworm strains with diverse resistance.

关键词： Silkworm(Bombyx mori L.) Fluoride-resistance capability Intestinal environment Intestinal microbiota Illumina Miseq

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Characterization and function of a short peptidoglycan recognition protein from the Chinese oak silkworm, Antheraea pernyi

Characterization and function of a short peptidoglycan recog...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Li-Shang Dai Cen Qian LeiWang Guo-Qing Wei Qiu-Ning Liu Yu Sun Cong-Fen Zhang Jun Li Dong-Ran Liu Bao-Jian Zhu Chao-Liang Liu College of Life Science Anhui Agricultural University

Peptidoglycan recognition proteins（PGRPs） are conserved proteins in animals from insects to mammals and play an important role in immune response by recognizing peptidoglycan on microbial surfaces. In this study, a PGRP gene from Antheraea pernyi, named Ap-PGRP-A, was isolated and characterized. Sequence analysis revealed that the full-length c DNA of Ap-PGRP-A was 961 bp, containing a 40 bp 5′-untranslated sequence, a 339 bp 3′-untranslated region and an open reading frame of 582 bp. This gene encoded a putative protein of 193 amino acid residues. Phylogenetic analysis indicated that Ap-PGRP-A had the closest protein sequence similarity to Antheraea mylitta PGRP. The recombinant protein was successfully expressed in Escherichia coli cells and a rabbit anti-Ap-PGRP-A antibody was also prepared. Real-time quantitative reverse transcription-PCR analysis showed that Ap-PGRP-A was extensively expressed in the fat body, midgut, hemocyte, malpighian tubule and epidermis of A. pernyi, especially in the fat body and midgut. The expression levels of Ap-PGRP-A were significantly up-regulated by three types of microorganisms, and Ap-PGRP-A expression was more sensitive in response to the Gram-negative bacterium E. coli than the Gram-positive bacterium Bacillus subtilis or the fungus Beauveria bassiana. These data indicate that Ap-PGRP-A may play a role in the innate immune responses of A. pernyi.

关键词： Antheraea pernyi Peptidoglycan recognition protein Expression Immune response

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RNAi silencing of the SoxE gene suppresses cell proliferation in silkworm BmN4 cells

RNAi silencing of the SoxE gene suppresses cell proliferatio...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Ling Wei Zhiqing Li Daojun Cheng Takahiro Kusakabe Minhui Pan Jun Duan Yonghu Wang Cheng Lu State Key Laboratory of Silkworm Genome Biology Southwest University School of Life Science Southwest University Laboratory of Silkworm Science Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences

The transcription factor SoxE is mainly expressed in the gonad and involved in the regulation of gonad development and sex determination in animals. Here, we used the silkworm ovary-derived BmN4-SID1 cell line to survey the roles of the silkworm SoxE protein(BmSoxE) and predict its candidate binding targets. RNAi-mediated silencing of BmSoxE expression suppressed cell proliferation in BmN4-SID1 cells. A further cell cycle analysis revealed that this inhibition of cell proliferation was largely due to cell cycle arrest in G1 phase when BmSox E expression was blocked in BmN4-SID1 cells. Genome-wide microarray expression analyses demonstrated that the expression levels of a set of genes were significantly altered following BmSox E RNAi. More than half of these genes contained conserved binding sites for HMG box domain of the Sox proteins and were predicted to be candidate binding targets for BmSoxE. Importantly, some of the candidate targets may be associated with the effect of BmSox E on cell proliferation. Several candidate target genes showed gonad-specific expression in silkworm larvae. Taken together, these data demonstrate that BmSoxE is required for cell proliferation in silkworm BmN4-SID1 cells and provide valuable information for further investigations of the molecular control exerted by the BmSoxE protein over cell proliferation and gonad development in the silkworm.

关键词： Silkworm SoxE RNAi Cell proliferation Target

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The silkworm GSTe4 is sensitive to phoxim and protects HEK293 cells against UV-induced cell apoptosis

The silkworm GSTe4 is sensitive to phoxim and protects HEK29...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： X.Y.Chen J.Liu C.D.Zhang Y.F.Li T.H.Liu L.Wang Q.Y.Yu Y.H.Zhang C.Lu M.H.Pan State Key Laboratory of Silkworm Genome Biology Southwest University The management of experimental teaching center ChongQing Medical University Department of Biochemistry and Molecular Biology ChongQing Medical University The Institute of Agricultural and Life Sciences Chongqing University The Sericultural Research Institute Sichuan Academy of Agricultural Science Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry Southwest University

Glutathione S-transferases（GSTs, EC 2.5.1.1 8） are a family of super enzymes with multiple functions that play a major role in the detoxification of endogenous and xenobiotic compounds. In our previous study, we have predicted 23 putative cytosolic GSTs in the silkworm genome using bioinformatic methods. In this study, we cloned and studied the insect-specific epsilon-class GST gene GSTe4 from the silkworm, Bombyx mori. The recombinant Bm GSTe4（Bac-Bm GSTe4） was overexpressed in SF- 9 cell lines, and it was found to have effective GST activity. We also found that the expression of Bm GSTe4 was especially down-regulated after the silkworms were fumigated with or ingested phoxim. Moreover, Bm GSTe4 protected HEK293 cells against UV-induced cell apoptosis. These results demonstrated that Bm GSTe4 has GST activity, is sensitive to phoxim, and plays a role in inhibition of UV-induced cell apoptosis.

关键词： glutathione S-transferase function sensitive to phoxim cell apoptosis

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An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori

An efficient strategy for producing a stable, replaceable, h...

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第十二届家（柞）蚕遗传育种暨良种繁育学术研讨会

作者： Long D Lu W Zhang Y Bi L Xiang Z Zhao A State Key Laboratory of Silkworm Genome Biology Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry Southwest University Guangxi Research Academy of Seficultural Science

We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm(Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-Rg G2, with up to 16%(w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the att P-flanked optimized fibroin H-chain expression cassette in its genome. A phi C31/att-system-based recombinase-mediated cassette exchange(RMCE) method could be used to integrate other genes of interest into the same genome locus between the att P sites in TS3-g2. Controlling for position effects with phi C31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs.

关键词： An efficient strategy for producing a stable Bombyx mori highly efficient transgene expression system in silkworm replaceable

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