Publications by authors named "Chengxiang Hou"

22 Publications

  • Page 1 of 1

Transcriptome reveal the response to Cry1Ac toxin in susceptible Bombyx mori.

Arch Insect Biochem Physiol 2021 May 5:e21794. Epub 2021 May 5.

Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China.

Bombyx mori as a representative in Lepidoptera is an important economic insect in agriculture production. Bacillus thuringiensis (Bt) is a bacterial pathogen in silkworm production. Understanding how silkworm respond to Bt-toxin can provide guidance to cultivate resistant silkworm strains. Cry1Ac is one type of Bt-toxin. In current research, Dazao, a susceptible B. mori strain to Bt-toxin, was treated by Cry1Ac toxin and compared its transcriptome with untreated samples. This analysis detected 1234 differentially expressed genes (DEGs). Gene Ontology, KEGG, and UniProt keyword enrichment analysis showed that DEGs include ATP-binding cassette (ABC) transporter, stress response, cuticle, and protein synthesis, and folding process. Five ABC genes were upregulated after Cry1Ac treatment including ABCA2, ABCA3, and ABCC4. They are also known as the transporters of Bt-toxin in lepidopteran insect. Expression of cuticle proteins was significantly increased at 6 h after Cry1Ac treatment. Sex-specific storage-proteins and heat shock protein were also upregulated in Cry1Ac treated samples. Our data provide an expression profile about the response of Cry1Ac toxin in susceptible B. mori strain.
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http://dx.doi.org/10.1002/arch.21794DOI Listing
May 2021

iTRAQ-based quantitative proteomic analysis of silkworm infected with Beauveria bassiana.

Mol Immunol 2021 Jul 28;135:204-216. Epub 2021 Apr 28.

School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212018, China; Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, 212018, China. Electronic address:

Beauveria bassiana is a harmful pathogen to the economically important insect silkworm, always causes serious disease to the silkworm, which results in great losses to the sericulture industry. In order to explore the silkworm (Bombyx mori) response to B. bassiana infection, differential proteomes of the silkworm responsive to B. bassiana infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) at the different stage of the 3rd instar silkworm larvae. Among the 5040 proteins identified with confidence level of ≥95 %, total 937 proteins were differentially expressed, of which 488 proteins were up-regulated and 449 proteins were down-regulated. 23, 15, 250, 649 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the B. bassiana infected larvae at 18, 24, 36, 48 h post infection (hpi) respectively. Based on GO annotations, 6, 4, 128, 316 DEPs were involved in biological processes, 12, 5, 143, 376 DEPs were involved in molecular functions, and 6, 3, 108, 256 DEPs were involved in cell components at 18, 24, 36, 48 hpi respectively. KEGG pathway analysis displayed that 18, 12, 210, 548 DEPs separately participated in 63, 35, 201, 264 signal transduction pathways at different time of infection, and moreover a higher proportion of DEPs involved in metabolic pathways. The cluster analysis on the DEPs of different infection stages distinguished a co-regulated DEP, lysozyme precursor, which was up-regulated at both the mRNA level and the protein level, indicating that the lysozyme protein kept playing an important role in defending the silkworm against B. bassiana infection. This was the first report using an iTRAQ approach to analyze proteomes of the whole silkworm against B. bassiana infection, which contributes to better understanding the defense mechanisms of silkworm to B. bassiana infection and provides important experimental data for the identification of key factors involved in the interaction between the pathogenic fungus and its host.
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http://dx.doi.org/10.1016/j.molimm.2021.04.018DOI Listing
July 2021

Inhibitory effects of Bombyx mori antimicrobial peptide cecropins on esophageal cancer cells.

Eur J Pharmacol 2020 Nov 4;887:173434. Epub 2020 Aug 4.

School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China. Electronic address:

Bombyx mori antimicrobial peptides (BmAMPs) are important effectors in silkworm immune system. They can inhibit and kill a variety of bacteria and fungi. Recent studies have shown that some kinds of BmAMPs exert strong inhibitory effects on a variety of tumor cells. In the present study, the antitumor activity of BmAMP Cecropin A (BmCecA) and BmAMP Cecropin D (BmCecD) was investigated against human esophageal cancer cells and their antitumor mechanism preliminary explored. Cell Counting Kit-8 and colony formation assays indicated that BmCecA and BmCecD suppressed cell proliferation and reduced colony formation of both Eca109 and TE13 cells in a dose-dependent manner, but exhibited no inhibitory effect on normal human embryonic kidney 293T cells. Wound healing and invasion experiments indicated that both BmCecA and BmCecD inhibited migration and invasion of Eca109 and TE13 cells in vitro. Annexin V/propidium iodide staining and flow cytometry detection suggested that BmCecA induced the apoptosis of Eca109 cells in a dose-dependent manner. RT-qPCR and western blot analysis showed that BmCecA induced apoptosis of Eca109 cells through the activation of a mitochondria-mediated caspase pathway, the upregulation of B-cell lymphoma 2 (Bcl-2)-associated X protein and the downregulation of Bcl-2. In addition, BmCecA significantly inhibited the growth of xenograft tumors in Eca109-bearing mice. These results suggested that BmCecA and BmCecD might serve as potential therapeutic agents for the treatment of cancer in the future.
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http://dx.doi.org/10.1016/j.ejphar.2020.173434DOI Listing
November 2020

Analysis of reassortant and intragenic recombination in Cypovirus.

Virol J 2020 04 6;17(1):48. Epub 2020 Apr 6.

School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212018, Jiangsu, China.

Cypoviruses (CPVs) are RNA viruses with segmented double-stranded genome and major pathogens of various insects, including economic insects like silkworms and pest insects for agricultural crops and forests. Genome reassortment and recombination are common phenomenon for viruses as a mechanism to expand host range and increase virulence. In the present study, we analyzed the reassortant and recombination events for CPVs. The results showed that two genome segments (S1 and S4) of BmCPV1-YN shared higher nucleotide identity with the corresponding segment of BmCPV1-I while others were all more closely to BmCPV1-SZ, suggesting BmCPV1-YN was originated from reassortant events between BmCPV1-I and BmCPV1-SZ. Recombination analyses revealed that S6 of BmCPV1-YN was a recombinant segment derived from BmCPV1-I and BmCPV1-SZ, and S10 of DpCPV1 was a recombinant segment emerged from BmCPV1-I and LdCPV1. Our findings provide the evidence for the fact that CPVs could undergo reassortant and recombinant events and enrich the knowledge about etiology and molecular epidemiology of CPVs.
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http://dx.doi.org/10.1186/s12985-020-01321-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7132967PMC
April 2020

Label-free LC-MS/MS proteomic analysis of the hemolymph of silkworm larvae infected with Beauveria bassiana.

J Invertebr Pathol 2019 09 3;166:107227. Epub 2019 Aug 3.

Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, China. Electronic address:

Beauveria bassiana, a pathogen of the economically important silkworm (Bombyx mori), causes serious losses in the sericulture industry; however, the mechanisms underlying B. bassiana infection and the silkworm response are not fully understood. To obtain new insights into the interaction between B. bassiana and its host, hemolymph samples from fifth instar silkworm larvae infected with B. bassiana were analyzed at 36-h post-inoculation using a label-free LC-MS/MS proteomic technique. In total, 671 proteins were identified in the hemolymph, including 87 differentially expressed proteins, 42 up-regulated and 45 down-regulated in infected larvae. Six were detected only in infected larvae, and five were detected only in uninfected larvae. Based on GO annotations, 48 of the differentially expressed proteins were involved in molecular functions, 42 were involved in biological processes, and 39 were involved in cell components. A KEGG pathway analysis indicated that these differentially expressed proteins participate in 85 signal transduction pathways, including the amoebiasis, MAPK signaling, Hippo signaling, Toll and Imd signaling, and lysosome pathways. The silkworm hemolymph is the main site for B. bassiana replication. We identified differentially expressed proteins involved in the regulation of the host response to B. bassiana infection, providing important experimental data for the identification of key factors contributing to the interaction between the pathogenic fungus and its host.
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http://dx.doi.org/10.1016/j.jip.2019.107227DOI Listing
September 2019

Physiological and Transcriptomic Changes during the Early Phases of Adventitious Root Formation in Mulberry Stem Hardwood Cuttings.

Int J Mol Sci 2019 Jul 29;20(15). Epub 2019 Jul 29.

College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212003, China.

The initiation and induction of root primordia are of great importance for adventitious root (AR) formation in cutting propagation of horticultural and forestry crops. However, the underlying mechanisms orchestrating these early phases of AR formation remain largely unexplored. Here, we investigated the physiological and transcriptomic changes during the early AR phases in mulberry stem hardwood cuttings. The results showed that the concentrations of soluble proteins increased, whereas concentrations of soluble sugars and starch were decreased. Indole-3-acetic acid (IAA) and zeatin had a rapid transit peak at 6 h after planting (hAP) and declined thereafter. The activities of peroxidase and catalase persistently increased and indole-3-acetic acid oxidase was maintained at a higher stable level from 0 hAP, while the activities of polyphenol oxidase fluctuated with soluble phenolics and IAA levels. The comparative transcriptome identified 4276 common genes that were differentially regulated at -6, 0 and 54 hAP. They were separated into five clusters with distinct biological functions such as defense response and photosynthesis. Considerable common genes were assigned to pathways of sugar metabolism, mitogen-activated protein kinase, and circadian rhythm. The gene co-expression network analysis revealed three major co-expressed modules involved in stress responses, hormone signaling, energy metabolism, starch metabolism, and circadian rhythm. These findings demonstrate the positive effect of auxin on AR induction, and uncovered the crucial roles of stress responses, hormone signaling and circadian rhythm in coordinating the physiological changes during the early phases of AR formation in mulberry stem hardwood cuttings.
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http://dx.doi.org/10.3390/ijms20153707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6696018PMC
July 2019

Expressional analysis of the silkworm storage protein 1 and identification of its interacting proteins.

Insect Mol Biol 2020 02 30;29(1):66-76. Epub 2019 Jul 30.

School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, China.

Storage proteins are haemolymph-specific proteins in insects, mainly synthesized in the fat body, released into the haemolymph, and then selectively reabsorbed by the fat body before pupation. These storage proteins play an important role in insect metamorphosis and egg development. Some of these storage proteins are responsive to pathogen infection and can even suppress pathogen multiplication. However, the mechanisms of the physiological, biochemical and immune-responsive functions of storage proteins remain unclear. In this study, the expression patterns of Bombyx mori storage protein 1 (BmSP1) during the larval stage were analysed. Then, BmSP1 protein fused with enhanced green fluorescent protein (EGFP) was successfully expressed in a B. mori baculovirus vector expression system. Quantitative real-time PCR showed that the expression level of BmSP1 increased with the advance of instars and reached the highest level in the fifth instar, especially in the fat body. Recombinant BmSP1 expressed in silkworm larvae inhibited haemolymph melanization. Then, proteins that interact with BmSP1 were identified with EGFP used as an antigenic determinant by co-immunoprecipitation. A 30 kDa low molecular weight lipoprotein PBMHP-6 precursor (BmLP6) was shown to interact with BmSP1. Yeast two-hybrid experiments confirmed the interaction between BmSP1 and BmLP6. The results obtained in this study will be helpful for further study of the functions of BmSP1 and BmLP6 in the regulatory network of silkworm development and innate immunity.
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http://dx.doi.org/10.1111/imb.12610DOI Listing
February 2020

Silkworm storage protein Bm30K-19G1 has a certain antifungal effects on Beauveria bassiana.

J Invertebr Pathol 2019 05 27;163:34-42. Epub 2019 Feb 27.

School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212018, China; Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China. Electronic address:

Storage proteins in the 30 K family are ubiquitous in the hemolymph of insects and play important roles in adult metamorphosis, development, egg formation, carrier transport and even host immunity. Some studies have shown that the 30 K proteins can inhibit apoptosis and have certain antifungal effects. The silkworm protein Bm30K-19G1 is a low molecular weight apolipoprotein that is abundant in hemolymph of fifth instar larvae. Our previous transcriptome sequencing, real-time PCR analysis and proteomic studies showed that the expression level of the 30 K protein gene was significantly up-regulated in the silkworm infected with Beauveria bassiana. In this study, the ORF sequence of Bm30K-19G1 was amplified by PCR. The sequence is 1311 bp in length and encodes a 436 amino acid peptide. Bm30K-19G1 was expressed in all tested tissues of fifth instar larvae, with highest expression in fat body and the lowest expression in the midgut. Bm30K-19G1 protein was successfully expressed in the prokaryotic expression system using pET-28a(+) as vector and E. coli Arctic Express (DE3) as the expression bacterium strain. The expressed recombinant Bm30K-19G1 protein has an inhibitory effect on the conidial germination and hyphal growth of B. bassiana. Bm30K-19G1 also inhibited the growth and reproduction of B. bassiana in vivo; the median lethal time of infected silkworms was postponed by 6.4 h and the time for death of all infected larvae was postponed by 10 h. The results indicated that the silkworm storage protein 30K-19G1 is an antifungal protein against B. bassiana and help to elucidate the molecular mechanism of silkworm resistance against B. bassiana.
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http://dx.doi.org/10.1016/j.jip.2019.02.008DOI Listing
May 2019

MicroRNA Expression Analysis of Naked Silkworms.

J Econ Entomol 2018 12;111(6):2876-2883

Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China.

The silk gland (SG) is characterized by the synthesis and secretion of silk protein in the economically important silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). Nd and Nd-s are two fibroin-secretion-deficient silkworm mutants. MicroRNA (miRNA) plays an important role in many biological processes, such as cell proliferation, differentiation, and apoptosis. Using the Dazao silkworm as a control, we explored the miRNA expression profiles in the SGs of u02 (Nd) and u05 (Nd-s) to reveal the potential functions of miRNAs in silk protein expression and SG development. Here, we sequenced small RNA libraries made from the whole SGs of three strains. There are 260, 236, and 233 known miRNAs and 20, 18, and 18 potential new miRNAs identified from Dazao, u02, and u05, respectively. Fifty-three miRNAs are differentially expressed between Dazao and u02, 51 between Dazao and u05, and 16 between u02 and u05. Gene ontology/KEGG analyses show that most of the predicted target genes of differentially expressed miRNAs were assigned to functional categories involved in cell proliferation, organ development, and cellular compartment structures. The miRNA expression profile of naked silkworms will pave the way for the understanding of SG development and the regulation of silk protein expression.
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http://dx.doi.org/10.1093/jee/toy235DOI Listing
December 2018

MicroRNA profile of silk gland reveals different silk yields of three silkworm strains.

Gene 2018 May 9;653:1-9. Epub 2018 Feb 9.

Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China; The Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Science, Zhenjiang, Jiangsu 212018, China. Electronic address:

Silk proteins are synthesized and secreted by the silk gland. The differential gene expression in it leads to different silk yield among various silkworm strains. As crucial factors, microRNAs (miRNAs) regulate protein synthesis at post-transcriptional level in silk gland. MiRNAs expression level in the silk gland of three silkworm strains (Jingsong, Lan10 and Dazao) was analyzed and 33 differentially expressed miRNAs (DEMs) were discovered between JingSong (JS) and Lan10 (L10), 60 DEMs between JS and Dazao, 54 DEMs between L10 and Dazao respectively. The DEMs target genes were predicted combing with two different methods and their functions were annotated according to gene ontology. Our previous studies showed that a batch of genes related to silk yield were identified in JS and L10 strains by comparative transcriptome and quantitative trait loci (QTL) method. Thirteen DEMs whose target genes are related to protein biosynthesis processes were screened by combining with these researches. Twelve DEMs potentially regulate nineteen genes which exist in our QTL results. Six common DEMs potentially regulate the genes in both of previous results. Finally, five DEMs were selected to verify their expression levels between JS and L10 by qRT-PCR, which showed similar difference as the results of small RNA-sequencing. MiRNAs in the silk gland may directly affect silk protein biosynthesis in different silkworm strains. In current work, we identified a batch of DEMs which potentially regulate the genes related to silk yield. Further functionally study of these miRNAs will contribute to improve varieties and boost the silk yield. Our research provides a basis for studying these miRNAs and their functions in silk production.
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http://dx.doi.org/10.1016/j.gene.2018.02.019DOI Listing
May 2018

Corrigendum to "Molecular Cloning, Bioinformatic Analysis, and Expression of Lebocin 5 Gene Related to Infection".

Biomed Res Int 2017 22;2017:5168354. Epub 2017 Nov 22.

School of Biotechnology and Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, China.

[This corrects the article DOI: 10.1155/2017/9390803.].
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http://dx.doi.org/10.1155/2017/5168354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735606PMC
November 2017

Molecular Cloning, Bioinformatic Analysis, and Expression of Lebocin 5 Gene Related to Infection.

Biomed Res Int 2017 17;2017:9390803. Epub 2017 Jan 17.

School of Biotechnology and Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, China.

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, , by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by , the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm.
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http://dx.doi.org/10.1155/2017/9390803DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5282435PMC
February 2017

Transgenic Clustered Regularly Interspaced Short Palindromic Repeat/Cas9-Mediated Viral Gene Targeting for Antiviral Therapy of Bombyx mori Nucleopolyhedrovirus.

J Virol 2017 04 29;91(8). Epub 2017 Mar 29.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

We developed a novel antiviral strategy by combining transposon-based transgenesis and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system for the direct cleavage of Bombyx mori nucleopolyhedrovirus (BmNPV) genome DNA to promote virus clearance in silkworms. We demonstrate that transgenic silkworms constitutively expressing Cas9 and guide RNAs targeting the BmNPV () and genes effectively induce target-specific cleavage and subsequent mutagenesis, especially large (∼7-kbp) segment deletions in BmNPV genomes, and thus exhibit robust suppression of BmNPV proliferation. Transgenic animals exhibited higher and inheritable resistance to BmNPV infection than wild-type animals. Our approach will not only contribute to modern sericulture but also shed light on future antiviral therapy. Pathogen genome targeting has shown its potential in antiviral research. However, transgenic CRISPR/Cas9 system-mediated viral genome targeting has not been reported as an antiviral strategy in a natural animal host of a virus. Our data provide an effective approach against BmNPV infection in a real-world biological system and demonstrate the potential of transgenic CRISPR/Cas9 systems in antiviral research in other species.
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http://dx.doi.org/10.1128/JVI.02465-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5375672PMC
April 2017

Expression profiling of Bombyx mori gloverin2 gene and its synergistic antifungal effect with cecropin A against Beauveria bassiana.

Gene 2017 Feb 9;600:55-63. Epub 2016 Nov 9.

Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, China; Chinese Academy of Agricultural Sciences, Zhenjiang 212018, China. Electronic address:

Gloverin2 is a cationic and glycine-rich antimicrobial peptide whose expression can be induced in fat body of silkworm (Bombyx mori) larvae exposed to bacteria. The purpose of this study is to identify the roles of Bombyx mori gloverin2 (Bmgloverin2) during entomopathogenic fungus Beauveria bassiana infection. Fluorescent quantitative real-time PCR analysis indicated that the relative expression level of Bmgloverin2 gene was up-regulated in the silkworm larvae infected by B. bassiana. The cDNA of Bmgloverin2 was cloned from the silkworm by RT-PCR and the DNA segment of the Bmgloverin2 peptide (without signal peptide sequence) was inserted into pCzn1 expression plasmid and expressed in E. coli ArcticExpress (DE3). SDS-PAGE results revealed that soluble recombinant Bmgloverin2 was successfully expressed and purified. Polyclonal antibody against the Bmgloverin2 was successfully produced with the expressed recombinant protein. Western blot analysis indicated that Bmgloverin2 could be detected in the fat body of silkworm larvae infected with B. bassiana, suggesting that the expression of Bmgloverin2 could be induced by B. bassiana infection in silkworm. Antifungal assays indicated that the Bmgloverin2 had a synergistic antifungal activity with B. mori cecropin A (BmCecA) to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that Bmgloverin2 exhibits synergistic effect with BmCecA in antifungal activity against B. bassiana. The study demonstrates that Bmgloverin2 is an antifungal protein which plays an important role in synergistic antifungal activity with other antimicrobial peptide in silkworm.
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http://dx.doi.org/10.1016/j.gene.2016.11.011DOI Listing
February 2017

Comparative Transcriptome Analysis Reveals Different Silk Yields of Two Silkworm Strains.

PLoS One 2016 9;11(5):e0155329. Epub 2016 May 9.

School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang Jiangsu 212018, China.

Cocoon and silk yields are the most important characteristics of sericulture. However, few studies have examined the genes that modulate these features. Further studies of these genes will be useful for improving the products of sericulture. JingSong (JS) and Lan10 (L10) are two strains having significantly different cocoon and silk yields. In the current study, RNA-Seq and quantitative polymerase chain reaction (qPCR) were performed on both strains in order to determine divergence of the silk gland, which controls silk biosynthesis in silkworms. Compared with L10, JS had 1375 differentially expressed genes (DEGs; 738 up-regulated genes and 673 down-regulated genes). Nine enriched gene ontology (GO) terms were identified by GO enrichment analysis based on these DEGs. KEGG enrichment analysis results showed that the DEGs were enriched in three pathways, which were mainly associated with the processing and biosynthesis of proteins. The representative genes in the enrichment pathways and ten significant DEGs were further verified by qPCR, the results of which were consistent with the RNA-Seq data. Our study has revealed differences in silk glands between the two silkworm strains and provides a perspective for understanding the molecular mechanisms determining silk yield.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155329PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861282PMC
July 2017

Bombyx mori cecropin A has a high antifungal activity to entomopathogenic fungus Beauveria bassiana.

Gene 2016 May 2;583(1):29-35. Epub 2016 Mar 2.

Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu, China; Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, Jiangsu, China. Electronic address:

A cDNA encoding cecropin A (CecA) was cloned from the larvae of silkworm, Bombyx mori, using RT-PCR. It encodes a protein of 63 amino acids, containing a 22 amino acid signal peptide and a 37 amino acid mat peptide of functional domain. The CecA secondary structure contains two typical amphiphilic α-helices. Real-time qPCR analysis revealed that CecA was expressed in all the tissues tested, including cuticle, fat body, hemocytes, Malpighian tubule, midgut and silk gland in the silkworm larvae with the highest expression in the fat body and hemocytes. The gene expression of B. mori CecA was rapidly induced by Beauveria bassiana challenge and reached maximum levels at 36h after inoculation in third instar larvae. In the fifth instar larvae infected with B. bassiana, the relative expression level of CecA was upregulated in fat body and hemocytes, but not in cuticle, Malpighian tubule, midgut and silk gland. The cDNA segment of the CecA was inserted into the expression plasmid pET-30a(+) to construct a recombinant expression plasmid. Western blot results revealed that his-tagged fusion protein was successfully expressed and purified. Then the mat peptide of CecA was chemically synthesized with C-terminus amidation for in vivo antifungal assay and purity achieved 93.7%. Mass spectrometry and SDS-PAGE showed its molecular weight to be 4046.95Da. Antifungal assays indicated that the B. mori CecA had a high antifungal activity to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that the CecA is effective to inhibit B. bassiana inside the body of silkworm.
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http://dx.doi.org/10.1016/j.gene.2016.02.045DOI Listing
May 2016

Transcriptome analysis of silkworm, Bombyx mori, during early response to Beauveria bassiana challenges.

PLoS One 2014 11;9(3):e91189. Epub 2014 Mar 11.

Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu, China; Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu, China.

Host-pathogen interactions are complex processes and it is a central challenge to reveal these interactions. Fungal infection of silkworm, Bombyx mori, may induce a variety of responsive reaction. However, little is known about the molecular mechanism of silkworm immune response against the fungal infection. To obtain an overview of the interaction between silkworm and an entomopathogenic fungus Beauveria bassiana, Digital Gene Expression profiling, a tag based high-throughput transcriptome sequencing method, was employed to screen and identify differentially expressed genes (DEGs, FDR ≤ 0.001, ∣log2ratio∣ ≥ 1) of silkworm larvae during early response against B. bassiana infection. Total 1430 DEGs including 960 up-regulated and 470 down-regulated ones were identified, of which 627 DEGs can be classified into GO categories by Gene Ontology (GO) analysis. KEGG pathways analysis of these DEGs suggested that many biological processes, such as defense and response, signal transduction, phagocytosis, regulation of gene expression, RNA splicing, biosynthesis and metabolism, protein transport etc. were involved in the interaction between the silkworm and B. bassiana. A number of differentially expressed fungal genes were also identified by mapping the sequencing tags to B. bassiana genome. These results provided new insights to the molecular mechanism of silkworm immune response to B. bassiana infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091189PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949756PMC
May 2015

Differential gene expression in silkworm in response to Beauveria bassiana infection.

Gene 2011 Sep 1;484(1-2):35-41. Epub 2011 Jun 1.

Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu, China.

Host-pathogen interactions are complex processes, and revealing these interactions is challenging. Beauveria bassiana is a destructive pathogen to the economically beneficial silkworm, Bombyx mori, but is also a good pathogenic material for investigating insect responses to fungal infection. For better understanding of the molecular regulation of immune response and the interactive mechanism between the silkworm and B. bassiana, suppression subtractive hybridization was employed to identify differentially expressed genes in the pathogen-stimulated silkworm larvae. Complementary DNA libraries were constructed, in which 240 clones were sequenced. A total of 77 genes were found to be involved in the infection process, among which 55 were known genes and 22 were novel. Expression profiling of 6 genes by quantitative PCR showed that they were induced by fungal challenge. This study establishes the first step to understanding the molecular mechanisms by which silkworm responds to fungal infection.
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http://dx.doi.org/10.1016/j.gene.2011.05.023DOI Listing
September 2011

Effects of insect viruses and pesticides on glutathione S-transferase activity and gene expression in Bombyx mori.

J Econ Entomol 2009 Aug;102(4):1591-8

School of Biology and Environmental Engineering, Jiangsu University of Science and Technology, Zhenjiang 212018, China.

Glutathione S-transferase (GST) is a gene family generally associated with detoxification and plays an important role in detoxifying exogenous compounds. The silkworm Bombyx mori is an important economic animal for silk production. However, it is liable to infection by a number of viruses and chemical agents that can contaminate its food and growing environment. Here we conducted a comparative study of strain- and tissue-specific expressions of GST in the silkworm under infections by Bombyx mori nuclear polyhedrosis virus (BmNPV) and Bombyx mori densonucleosis virus (BmDNV) and under the stress of pesticides (insecticide and herbicide). BmDNV induced an increase of GST at 24 h and a decrease at 48 and 72 h in the midgut of the DNV-susceptible strain and a decrease at 24 h and increase at 48 and 72 h in the midgut of the DNV-tolerant strain. BmDNV induced a significant increase of GST from 24 to 72 h in the fat body of both DNV-susceptible and DNV-tolerant strains. In contrast, BmNPV induced a significant decrease of GST in both the fat body and midgut of the NPV-susceptible strain and induced increase of GST from 24 to 48 h in the midgut and at 72 h in the fat body of the NPV-tolerant strain. All of these results suggest that BmGST activity varies with the strain, tissue, feeding behavior, and developmental stage of the silkworm. On treating silkworm larvae with pesticides (insecticide and herbicide), expression of the BmGSTS2 gene increased noticeably in the midgut and reached a peak at 6 to 12 h. The mRNA level of BmGSTS2 in the midgut increased during administration of the chemicals, suggesting that the induction of BmGSTS2 is part of the defense mechanism against exogenous chemicals.
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http://dx.doi.org/10.1603/029.102.0425DOI Listing
August 2009

Analyzing genetic relationships in Bombyx mori using intersimple sequence repeat amplification.

J Econ Entomol 2007 Feb;100(1):202-8

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 200032, People's Republic of China.

Intersimple sequence repeat (ISSR) amplification was used to analyze genetic relationships among silkworm, Bombyx mori L., strains. Nineteen primers containing simple sequence repeat (SSR) motifs were tested for amplification on a panel of 42 strains, representative of the diversity of silkworm germplasm; 12 of the primers amplified distinct, reproducible bands. The primers amplified a total of 108 bands, of which 85 (78.7%) were polymorphic. The ISSR results suggested that within the dinucleotide class, the poly(CA) motif was more common than the poly(CT) motif. The ISSR amplification pattern was used to group the silkworm strains into seven subclusters based on their origin in an unweighted pair-group method with arithmetic average cluster analysis by using Nei's genetic distance. Seven major ecotypic silkworm groups were analyzed. Principal component analysis of the ISSR data supported the unweighted pair-group method with arithmetic average clustering. Therefore, ISSR amplification is a valuable method for determining genetic variability among silkworm varieties. This efficient genetic fingerprinting technique should be useful for characterizing the large numbers of silkworm strains held in national and international germplasm centers.
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http://dx.doi.org/10.1603/0022-0493(2007)100[202:agribm]2.0.co;2DOI Listing
February 2007

Linkage and mapping analyses of the densonucleosis non-susceptible gene nsd-Z in the silkworm Bombyx mori using SSR markers.

Genome 2006 Apr;49(4):397-402

Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018, PR China.

In the silkworm Bombyx mori, non-susceptibility to the Zhenjiang (China) strain of the densonucleosis virus (DNV-Z) is controlled by the recessive gene nsd-Z (non-susceptible to DNV-Z), which is located on chromosome 15. Owing to a lack of crossing over in females, reciprocal backcrossed F1 (BC1) progeny were used for linkage analysis and mapping of the nsd-Z gene using silkworm strains Js and L10, which are classified as being highly susceptible and non-susceptible to DNV-Z, respectively. BC1 larvae were inoculated with the DNV-Z virus at the first instar, and DNA was extracted from the individual surviving pupae and analyzed for simple sequence repeat (SSR) markers. The nsd-Z gene was found to be linked to 7 SSR markers, as all the surviving larvae in the BC1female (F1female x L10male) showed the homozygous profile of strain L10, and the sick larvae in the BC1female (F1female x L10male) showed the heterozygous profile of Js x L10 F1 hybrids. Using a reciprocal BC1male (L101female x F1male) cross, we constructed a linkage map of 80.6 cM, with nsd-Z mapped at 30 cM and the closest SSR marker at a distance of 4.4 cM.
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http://dx.doi.org/10.1139/g05-125DOI Listing
April 2006

Genetic diversity among silkworm (Bombyx mori L., Lep., Bombycidae) germplasms revealed by microsatellites.

Genome 2005 Oct;48(5):802-10

Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, P. R. China.

To determine genetic relationships among strains of silkworm, Bombyx mori L., 31 strains with different origins, number of generations per year, number of molts per generation, and morphological characters were studied using simple sequence repeat (SSR) markers. Twenty-six primer pairs flanking microsatellite sequences in the silkworm genome were assayed. All were polymorphic and unambiguously separated silkworm strains from each other. A total of 188 alleles were detected with a mean value of 7.2 alleles/locus (range 2-17). The average heterozygosity value for each SSR locus ranged from 0 to 0.60, and the highest one was 0.96 (Fl0516 in 4013). The mean polymorphism index content (PIC) was 0.66 (range 0.12-0.89). Unweighted pair group method with arithmetic means (UPGMA) cluster analysis of Nei's genetic distance grouped silkworm strains based on their origin. Seven major ecotypic silkworm groups were analyzed. Principal components analysis (PCA) for SSR data support their UPGMA clustering. The results indicated that SSR markers are an efficient tool for fingerprinting cultivars and conducting genetic-diversity studies in the silkworm.
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http://dx.doi.org/10.1139/g05-053DOI Listing
October 2005