Publications by authors named "Muwang Li"

32 Publications

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

Transcriptomic analysis at the first instar larval stage of nonmolting Bombyx mori mutant (a42).

Arch Insect Biochem Physiol 2020 May 19;104(1):e21663. Epub 2020 Feb 19.

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

The life cycle of the holometabolous insect Bombyx mori (Linnaeus) consists of the embryo, larva, pupa, and adult stages with six larval molts. Ecdysone and juvenile hormones play important roles in the growth and development of the silkworms. The a42 silkworm mutant is recessive and homozygous lethal by exhibiting a dark-colored and small body size and fails to molt to second instar. We compared the gene expression of a42 mutants with normal individuals at the first larval molting stage to elucidate the physiological influence of the a42 mutation on the growth and development of silkworms. The transcriptomic sequencing results revealed that 1,411 genes are differentially expressed in a42 mutants, compared with wild-type control silkworms, in which 791 genes are upregulated and 620 genes are downregulated. Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analyses identified differentially expressed genes (DEGs) assigned to biological pathways, such as pentose and glucoronate interconversions, glycerolipid metabolism, folate biosynthesis, amino sugar, and nucleotide sugar metabolism. Two hydroxylases of phenylalanine hydroxylase (BmPAH) and tyrosine hydroxylase (BmTh) are upregulated in a42 mutants. The influence of a42 mutation on these DEGs reveals that melanin metabolism plays an important role during the molting process in silkworms.
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http://dx.doi.org/10.1002/arch.21663DOI Listing
May 2020

The silkworm (Bombyx mori) neuropeptide orcokinin is involved in the regulation of pigmentation.

Insect Biochem Mol Biol 2019 11 23;114:103229. Epub 2019 Aug 23.

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

The natural colorful cuticles of insects play important roles in many physiological processes. Pigmentation is a physiological process with a complex regulatory network whose regulatory mechanism remains unclear. Bombyx mori pigmentation mutants are ideal materials for research on pigmentation mechanisms. The purple quail-like (q-l) and brown quail-like (q-l) mutants originated from plain silkworm breeds 932VR and 0223JH respectively exhibit similar cuticle pigmentation to that of the quail mutant. The q-l mutant also presents a developmental abnormality. In this study, genes controlling q-l and q-l mutants were located on chromosome 8 by positional cloning. Then the neuropeptide gene orcokinin (OK) was identified to be the major gene responsible for two quail-like mutants. The B. mori orcokinin gene (BommoOK) produces two transcripts, BommoOKA and BommoOKB, by alternative splicing. The CRISPR/Cas9 system and orcokinin peptides injection were used for further functional verification. We show a novel function of BommoOKA in inhibiting pigmentation, and one mature peptide of orcokinin A, OKA_type2, is the key factor in pigmentation inhibition. These results provide a reference for studying the function of orcokinin and are of theoretical importance for studying the regulatory mechanism of pigmentation.
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http://dx.doi.org/10.1016/j.ibmb.2019.103229DOI Listing
November 2019

Alternative splicing regulation of doublesex gene by RNA-binding proteins in the silkworm Bombyx mori.

RNA Biol 2019 06 18;16(6):809-820. Epub 2019 Mar 18.

c College of Life Science , Wuhan University , Wuhan , China.

Doublesex is highly conserved and sex-specifically spliced in insect sex-determination pathways, and its alternative splicing (AS) is regulated by Transformer, an exonic splicing activator, in the model system of Drosophila melanogaster. However, due to the lack of a transformer gene, AS regulation of doublesex remains unclear in Lepidoptera, which contain the economically important silkworm Bombyx mori and thousands of agricultural pests. Here, we use yeast three-hybrid system to screen for RNA-binding proteins that recognize sex-specific exons 3 and 4 of silkworm doublesex (Bm-dsx); this approach identified BxRBP1/Lark binding to the exon 3, and BxRBP2/TBPH and BxRBP3/Aret binding to the exon 4. Investigation of tissues shows that BxRBP1 and BxRBP2 have no sex specificity, but BxRBP3 has - three of its four isoforms are expressed with a sex-bias. Using novel sex-specific silkworm cell lines, we find that BxRBP1 and BxRBP3 directly interact with each other, and cooperatively function as splicing repressors. Over-expression of BxRBP1 and BxRBP3 isoforms efficiently inhibits splicing of the exons 3 and 4 in the female-specific cells and generates the male-specific isoform of Bm-dsx. We also demonstrate that the sex-determination upstream gene Masc regulates alternatively transcribed BxRBP3 isoforms. Thus, we identify a new regulatory mechanism of doublesex AS in the silkworm, revealing an evolutionary divergence in insect sex-determination.
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http://dx.doi.org/10.1080/15476286.2019.1590177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546364PMC
June 2019

Proteomic Analysis of Larval Integument in a Dominant Obese Translucent (Obs) Silkworm Mutant.

J Insect Sci 2018 Nov 1;18(6). Epub 2018 Nov 1.

State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University, Tiansheng Road, Beibei, Chongqing, China.

The dominant obese translucent (Obs) mutant of the silkworm (Bombyx mori) results in a short and stout larval body, translucent phenotype, and abnormal pigmentation in the integument. The Obs mutant also displays deficiency in ecdysis and metamorphosis. In the present study, to gain an understanding of multiple Obs phenotypes, we investigated the phenotypes of Obs and performed a comparative analysis of the larval integument proteomes of Obs and normal silkworms. The phenotypic analysis revealed that the Obs larvae were indeed short and fat, and that chitin and uric acid content were lower but melanin content was higher in the Obs mutant. Proteomic analysis revealed that 244 proteins were significantly differentially expressed between Obs and normal silkworms, some of which were involved in uric acid metabolism and melanin pigmentation. Twenty-six proteins were annotated as cuticular proteins, including RR motif-rich cuticular proteins (CPR), glycine-rich cuticular protein (CPG), hypothetical cuticular protein (CPH), cuticular protein analogous to peritrophins (CPAPs), and the chitin_bind_3 motif proteins, and accounted for over 84% of the abundance of the total significantly differentially expressed proteins. Moreover, 22 of the 26 cuticular proteins were downregulated in the Obs mutant. Comparative proteomic analysis suggested that the multiple phenotypes of the Obs mutant might be related to changes in the expression of proteins that participate in cuticular formation, uric acid metabolism, and melanin pigmentation. These results could lay a basis for further identification of the gene responsible for the Obs mutant. The data have been deposited to ProteomeXchange with identifier PXD010998.
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http://dx.doi.org/10.1093/jisesa/iey098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225826PMC
November 2018

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

Silkworm genetic sexing through W chromosome-linked, targeted gene integration.

Proc Natl Acad Sci U S A 2018 08 13;115(35):8752-8756. Epub 2018 Aug 13.

Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032 Shanghai, China;

Sex separation methods are critical for genetic sexing systems in commercial insect production and sterile insect techniques. Integration of selectable marker genes into a sex chromosome is particularly useful in insects with a heterogametic sex determination system. Here, we describe targeted gene integration of fluorescent marker expression cassettes into a randomly amplified polymorphic DNA (RAPD) marker region in the W chromosome of the lepidopteran model insect using transcriptional activator-like effector nuclease (TALEN)-mediated genome editing. This silkworm strain shows ubiquitous female-specific red or green fluorescence from the embryonic to adult stages. Furthermore, we developed a binary, female-specific, embryonic lethality system combining the TALEN and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology. This system includes one strain with TALEN-mediated, W-specific Cas9 expression driven by the silkworm germ cell-specific () promoter and another strain with U6-derived single-guide RNA (sgRNA) expression targeting (), an essential gene for silkworm embryonic development. Filial 1 (F1) hybrids exhibit complete female-specific lethality during embryonic stages. Our study provides a promising approach for genetic sexing and sheds light on developing sterile insect techniques in other insect species, especially in lepidopteran pests with WZ/ZZ sex chromosome systems.
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http://dx.doi.org/10.1073/pnas.1810945115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126770PMC
August 2018

Mass spider silk production through targeted gene replacement in .

Proc Natl Acad Sci U S A 2018 08 6;115(35):8757-8762. Epub 2018 Aug 6.

Key Laboratory of Insect Developmental and Evolutionary Biology, Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 200032 Shanghai, China;

Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, , has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene () with the major ampullate spidroin-1 gene () in the spider We successfully replaced the ∼16-kb endogenous gene with a 1.6-kb gene fused with a 1.1-kb partial sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.
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http://dx.doi.org/10.1073/pnas.1806805115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126722PMC
August 2018

The evolutionary road from wild moth to domestic silkworm.

Nat Ecol Evol 2018 08 2;2(8):1268-1279. Epub 2018 Jul 2.

CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai, China.

The Silk Road, which derives its name from the trade of silk produced by the domestic silkworm Bombyx mori, was an important episode in the development and interaction of human civilizations. However, the detailed history behind silkworm domestication remains ambiguous, and little is known about the underlying genetics with respect to important aspects of its domestication. Here, we reconstruct the domestication processes and identify selective sweeps by sequencing 137 representative silkworm strains. The results present an evolutionary scenario in which silkworms may have been initially domesticated in China as trimoulting lines, then subjected to independent spreads along the Silk Road that gave rise to the development of most local strains, and further improved for modern silk production in Japan and China, having descended from diverse ancestral sources. We find that genes with key roles in nitrogen and amino acid metabolism may have contributed to the promotion of silk production, and that circadian-related genes are generally selected for their adaptation. We additionally identify associations between several candidate genes and important breeding traits, thereby advancing the applicable value of our resources.
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http://dx.doi.org/10.1038/s41559-018-0593-4DOI Listing
August 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

Comparative transcriptomes analysis of the wing disc between two silkworm strains with different size of wings.

PLoS One 2017 15;12(6):e0179560. Epub 2017 Jun 15.

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

Wings of Bombyx mori (B. mori) develop from the primordium, and different B. mori strains have different wing types. In order to identify the key factors influencing B. mori wing development, we chose strains P50 and U11, which are typical for normal wing and minute wing phenotypes, respectively. We dissected the wing disc on the 1st-day of wandering stage (P50D1 and U11D1), 2nd-day of wandering stage (P50D2 and U11D2), and 3rd-day of wandering stage (P50D3 and U11D3). Subsequently, RNA-sequencing (RNA-Seq) was performed on both strains in order to construct their gene expression profiles. P50 exhibited 628 genes differentially expressed to U11, 324 up-regulated genes, and 304 down-regulated genes. Five enriched gene ontology (GO) terms were identified by GO enrichment analysis based on these differentially expressed genes (DEGs). KEGG enrichment analysis results showed that the DEGs were enriched in five pathways; of these, we identified three pathways related to the development of wings. The three pathways include amino sugar and nucleotide sugar metabolism pathway, proteasome signaling pathway, and the Hippo signaling pathway. The representative genes in the enrichment pathways were further verified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The RNA-Seq and qRT-PCR results were largely consistent with each other. Our results also revealed that the significantly different genes obtained in our study might be involved in the development of the size of B. mori wings. In addition, several KEGG enriched pathways might be involved in the regulation of the pathways of wing formation. These results provide a basis for further research of wing development in B. mori.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179560PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5472328PMC
September 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

Depletion of juvenile hormone esterase extends larval growth in Bombyx mori.

Insect Biochem Mol Biol 2017 02 3;81:72-79. Epub 2017 Jan 3.

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production.
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http://dx.doi.org/10.1016/j.ibmb.2017.01.001DOI 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

Mutation of a Cuticle Protein Gene, BmCPG10, Is Responsible for Silkworm Non-Moulting in the 2nd Instar Mutant.

PLoS One 2016 20;11(4):e0153549. Epub 2016 Apr 20.

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

In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2nd instar (nm2). Genetic analysis indicated that the nm2 mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, nm2 was located in a region approximately 275 kb on the 5th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the BmCPG10 gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene's structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The BmCPG10 mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The BmCPG10 mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of nm2 larvae was lower than that of the wild-type larvae. The nm2 mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7-dehydrocholesterol (7dC), but the rescued nm2 only developed to the 4th instar and subsequently died. The moulting time of silkworms could be delayed by BmCPG10 RNAi. Thus, we speculated that the mutation of BmCPG10 was responsible for the silkworm mutant that did not moult in the 2nd instar.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153549PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838254PMC
September 2016

Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system.

J Insect Physiol 2015 Aug 9;79:73-9. Epub 2015 Jun 9.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.
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http://dx.doi.org/10.1016/j.jinsphys.2015.06.004DOI Listing
August 2015

Allelic-specific expression in relation to Bombyx mori resistance to Bt toxin.

Insect Biochem Mol Biol 2014 Nov 12;54:53-60. Epub 2014 Aug 12.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

Understanding the mechanism of Bt resistance is one of the key elements of the effective application of Bt in pest control. The lepidopteran model insect, the silkworm, demonstrates qualities that make it an ideal species to use in achieving this understanding. We screened 45 strains of silkworm (Bombyx mori) using a Cry1Ab toxin variant. The sensitivity levels of the strains varied over a wide range. A resistant strain (P50) and a phylogenetically related susceptible strain (Dazao) were selected to profile the expressions of 12 Bt resistance-related genes. The SNPs in these genes were detected based on EST analysis and were validated by allelic-specific PCR. A comparison of allelic-specific expression between P50 and Dazao showed that the transcript levels of heterozygous genes containing two alleles rather than an imbalanced allelic expression contribute more to the resistance of P50 against Bt. The responses of the allelic-specific expression to Bt in hybrid larvae were then investigated. The results showed that the gene expression pattern of an ATP-binding cassette transporter C2 (ABCC2) and an aminopeptidase N (APN3), changed in an allelic-specific manner, with the increase of the resistant allele expression correlated with larval survival. The results suggest that a trans-regulatory mechanism in ABCC2 and APN3 allelic-specific expression is involved in the insect's response to the Bt toxin. The potential role of allelic-specific gene regulation in insect resistance to Bt toxins is discussed.
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http://dx.doi.org/10.1016/j.ibmb.2014.07.007DOI Listing
November 2014

Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

Arch Insect Biochem Physiol 2015 Jan 10;88(1):64-84. Epub 2014 Jul 10.

College of Life Science, Anhui Agricultural University, Changjiang, People's Republic of China; College of Agriculture and Biology, Shanghai Jiaotong University, Dongchuan, Shanghai, People's Republic of China; Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, People's Republic of China.

Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians.
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http://dx.doi.org/10.1002/arch.21178DOI Listing
January 2015

Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm, Bombyx mori.

Insect Biochem Mol Biol 2013 Jul 6;43(7):562-71. Epub 2013 Apr 6.

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

ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-ok demonstrated that repression of Bm-ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis.
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http://dx.doi.org/10.1016/j.ibmb.2013.03.011DOI Listing
July 2013

Reduced expression of the dysbindin-like gene in the Bombyx mori ov mutant exhibiting mottled translucency of the larval skin.

Genome 2013 Feb 14;56(2):101-8. Epub 2012 Dec 14.

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

The ov (mottled translucent of Var) mutant, an oily mutant of Bombyx mori, exhibits mottled translucent skin with a varying degree of transparency among individuals. By linkage analysis of 2112 backcross individuals using polymorphic DNA markers, we successfully mapped a 179-kb region of chromosome 20 responsible for the ov phenotype. This region contains nine predicted genes. We compared the mRNA expression of these nine genes between the wild type and mutants and found that the expression of one of them, Bmdysb, was strikingly decreased in the epidermis of ov as well as its allelomorph, ov(p). Moreover, its expression level was well correlated with the degree of transparency among individuals. Bmdysb was homologous to DTNBP1 encoding human dysbindin, a subunit of the biogenesis of lysosome-related organelles complex-1. Our results suggest that the translucent skin may be due to repression of Bmdysb in the ov mutants and that Bmdysb plays an important role in the formation and accumulation of urate granules in the silkworm epidermis.
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http://dx.doi.org/10.1139/gen-2012-0127DOI Listing
February 2013

Alternative splicing and trans-splicing events revealed by analysis of the Bombyx mori transcriptome.

RNA 2012 Jul 24;18(7):1395-407. Epub 2012 May 24.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. We identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is expressed sex-specifically in silkworm gonads. Trans-splicing of mod(mdg4) in silkworm was experimentally confirmed. We identified integrations from a common 5'-gene with 46 newly identified alternative 3'-exons that are located on both DNA strands over a 500-kb region. Other trans-splicing events in B. mori were predicted by bioinformatic analysis, in which 12 events were confirmed by RT-PCR, six events were further validated by chimeric SNPs, and two events were confirmed by allele-specific RT-PCR in F(1) hybrids from distinct silkworm lines of JS and L10, indicating that trans-splicing is more widespread in insects than previously thought. Analysis of the B. mori transcriptome by RNA-seq provides valuable information of regulatory alternative splicing events. The conservation of splicing events across species and newly identified trans-splicing events suggest that B. mori is a good model for future studies.
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http://dx.doi.org/10.1261/rna.029751.111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383970PMC
July 2012

Disruption of an N-acetyltransferase gene in the silkworm reveals a novel role in pigmentation.

Development 2010 Dec;137(23):4083-90

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.

The pigmentation of insects has served as an excellent model for the study of morphological trait evolution and developmental biology. The melanism (mln) mutant of the silkworm Bombyx mori is notable for its strong black coloration, phenotypic differences between larval and adult stages, and its widespread use in strain selection. Here, we report the genetic and molecular bases for the formation of the mln morphological trait. Fine mapping revealed that an arylalkylamine N-acetyltransferase (AANAT) gene co-segregates with the black coloration patterns. Coding sequence variations and expression profiles of AANAT are also associated with the melanic phenotypes. A 126 bp deletion in the mln genome causes two alternatively spliced transcripts with premature terminations. An enzymatic assay demonstrated the absolute loss of AANAT activity in the mutant proteins. We also performed RNA interference of AANAT in wild-type pupae and observed a significant proportion of adults with ectopic black coloration. These findings indicate that functional deletion of this AANAT gene accounts for the mln mutation in silkworm. AANAT is also involved in a parallel melanin synthesis pathway in which ebony plays a role, whereas no pigmentation defect has been reported in the Drosophila model or in other insects to date. To the best of our knowledge, the mln mutation is the first characterized mutant phenotype of insects with AANAT, and this result contributes to our understanding of dopamine metabolism and melanin pattern polymorphisms.
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http://dx.doi.org/10.1242/dev.053678DOI Listing
December 2010

Single base-resolution methylome of the silkworm reveals a sparse epigenomic map.

Nat Biotechnol 2010 May 2;28(5):516-20. Epub 2010 May 2.

CAS-Max Planck Junior Research Group, State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, China.

Epigenetic regulation in insects may have effects on diverse biological processes. Here we survey the methylome of a model insect, the silkworm Bombyx mori, at single-base resolution using Illumina high-throughput bisulfite sequencing (MethylC-Seq). We conservatively estimate that 0.11% of genomic cytosines are methylcytosines, all of which probably occur in CG dinucleotides. CG methylation is substantially enriched in gene bodies and is positively correlated with gene expression levels, suggesting it has a positive role in gene transcription. We find that transposable elements, promoters and ribosomal DNAs are hypomethylated, but in contrast, genomic loci matching small RNAs in gene bodies are densely methylated. This work contributes to our understanding of epigenetics in insects, and in contrast to previous studies of the highly methylated genomes of Arabidopsis and human, demonstrates a strategy for sequencing the epigenomes of organisms such as insects that have low levels of methylation.
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http://dx.doi.org/10.1038/nbt.1626DOI Listing
May 2010

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

An integrated genetic linkage map for silkworms with three parental combinations and its application to the mapping of single genes and QTL.

BMC Genomics 2009 Aug 21;10:389. Epub 2009 Aug 21.

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, PR China.

Background: Bombyx mori, the domesticated silkworm, is a well-studied model insect with great economic and scientific significance. Although more than 400 mutations have been described in silkworms, most have not been identified, especially those affecting economically-important traits. Simple sequence repeats (SSRs) are effective and economical tools for mapping traits and genetic improvement. The current SSR linkage map is of low density and contains few polymorphisms. The purpose of this work was to develop a dense and informative linkage map that would assist in the preliminary mapping and dissection of quantitative trait loci (QTL) in a variety of silkworm strains.

Results: Through an analysis of > 50,000 genotypes across new mapping populations, we constructed two new linkage maps covering 27 assigned chromosomes and merged the data with previously reported data sets. The integrated consensus map contains 692 unique SSR sites, improving the density from 6.3 cM in the previous map to 4.8 cM. We also developed 497 confirmed neighboring markers for corresponding low-polymorphism sites, with 244 having polymorphisms. Large-scale statistics on the SSR type were suggestive of highly efficient markers, based upon which we searched 16,462 available genomic scaffolds for SSR loci. With the newly constructed map, we mapped single-gene traits, the QTL of filaments, and a number of ribosomal protein genes.

Conclusion: The integrated map produced in this study is a highly efficient genetic tool for the high-throughput mapping of single genes and QTL. Compared to previous maps, the current map offers a greater number of markers and polymorphisms; thus, it may be used as a resource for marker-assisted breeding.
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http://dx.doi.org/10.1186/1471-2164-10-389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2741490PMC
August 2009

Insect-Specific microRNA Involved in the Development of the Silkworm Bombyx mori.

PLoS One 2009 5;4(3):e4677. Epub 2009 Mar 5.

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

MicroRNAs (miRNAs) are endogenous non-coding genes that participate in post-transcription regulation by either degrading mRNA or blocking its translation. It is considered to be very important in regulating insect development and metamorphosis. We conducted a large-scale screening for miRNA genes in the silkworm Bombyx mori using sequence-by-synthesis (SBS) deep sequencing of mixed RNAs from egg, larval, pupal, and adult stages. Of 2,227,930 SBS tags, 1,144,485 ranged from 17 to 25 nt, corresponding to 256,604 unique tags. Among these non-redundant tags, 95,184 were matched to the silkworm genome. We identified 3,750 miRNA candidate genes using a computational pipeline combining RNAfold and TripletSVM algorithms. We confirmed 354 miRNA genes using miRNA microarrays and then performed expression profile analysis on these miRNAs for all developmental stages. While 106 miRNAs were expressed in all stages, 248 miRNAs were egg- and pupa-specific, suggesting that insect miRNAs play a significant role in embryogenesis and metamorphosis. We selected eight miRNAs for quantitative RT-PCR analysis; six of these were consistent with our microarray results. In addition, we searched for orthologous miRNA genes in mammals, a nematode, and other insects and found that most silkworm miRNAs are conserved in insects, whereas only a small number of silkworm miRNAs has orthologs in mammals and the nematode. These results suggest that there are many miRNAs unique to insects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0004677PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650705PMC
June 2009

Molecular cloning and characterization of Bombyx mori CREB gene.

Arch Insect Biochem Physiol 2009 May;71(1):31-44

College of Life Sciences, Shanghai University, Shanghai, P.R. China, 200444.

The cAMP response element binding protein (CREB), as one of the best characterized stimulus-induced transcription factors, plays critical roles in activating transcription of target genes in response to a variety of environmental stimuli. To characterize this important molecule in the silkworm, Bombyx mori, we cloned a full-length cDNA of CREB gene from B. mori brains by using RACE-PCR. The sequence of B. mori CREB (named BmCREB1) gene contains a 88 bp 5' UTR, a 783 bp open reading frame (ORF) encoding 261 amino acids and a 348 bp 3' UTR. The deduced BmCREB amino acid sequence has 56.7% and 37.2% homology with CREB from Apis mellifera carnica and Drosophila melanogaster, respectively. The primary structure of the deduced BmCREB1 protein contains a kinase-inducible domain (KID) and a basic region/leucine zipper (bZIP) dimerization domain which exists in all CREB family members. Genomic analysis showed there are 9 exons and 5 introns in B. mori CREB genome sequences. We identified three different isoforms of BmCREB (BmCREB1, BmCREB2 and BmCREB3) through alternative splicing in C terminal. In addition, the expression of BmCREB in different developmental stages was investigated by using quantitative real-time PCR in both diapause and non-diapause type of B. mori bivoltine race (Dazao). BmCREB transcripts showed two peaks in embryonic stage and pupal stage in both types of bivoltine race. However, consistently higher expression of BmCREB was found throughout the developmental stages in the diapause type than in the non-diapause type. These results suggest that BmCREB is involved in the process of diapause induced by environmental factors.
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http://dx.doi.org/10.1002/arch.20292DOI Listing
May 2009

SAGE tag based cDNA microarray analysis during larval to pupal development and isolation of novel cDNAs in Bombyx mori.

Genomics 2007 Sep 20;90(3):372-9. Epub 2007 Jun 20.

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, People's Republic of China.

Many genes act together during the complex process of insect larval and pupal development. 20-hydroxyecdysone interacts with juvenile hormone to control insect growth and development and then activates several transcription factors, i.e., Broad, E74, and E75, and, subsequently, the late target genes. To investigate this phenomenon, we used serial analysis of gene expression (SAGE) tag-based cDNA microarray analysis to monitor the global gene expression profile during larval development and larva-pupa metamorphosis of the silkworm Bombyx mori. Of the 330 clones that were dotted to the chip, 267 were obtained by generating longer cDNA fragments from SAGE tags for gene identification, and the others were obtained from SAGE tag-matched genes or expressed sequence tags from public databases. According to the gene expression profile, the genes were classified into 12 clusters using a self-organizing map analysis. The results were partially confirmed using real-time reverse transcription-polymerase chain reaction. We obtained 22 full-length cDNAs using rapid amplification of 5' cDNA ends, of which eight genes were novel in the silkworm. Our results indicated that use of a cDNA microarray based on SAGE tags is effective for identifying and examining some low-expression genes associated with insect development.
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http://dx.doi.org/10.1016/j.ygeno.2007.05.005DOI Listing
September 2007

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