Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori

Insect chemosensory proteins (CSPs) as well as odorant-binding proteins (OBPs) have been supposed to transport hydrophobic chemicals to receptors on sensory neurons. Compared with OBPs, CSPs are expressed more broadly in various insect tissues. We performed a genome-wide analysis of the candidate CSP gene family in the silkworm. A total of 20 candidate CSPs, including 3 gene fragments and 2 pseudogenes, were characterized based on their conserved cysteine residues and their similarity to CSPs in other insects. Some of these genes were clustered in the silkworm genome. The gene expression pattern of these candidates was investigated using RT-PCR and microarray, and the results showed that these genes were expressed primarily in mature larvae and the adult moth, suggesting silkworm CSPs may be involved in development. The majority of silkworm CSP genes are expressed broadly in tissues including the antennae, head, thorax, legs, wings, epithelium, testes, ovaries, pheromone glands, wing disks, and compound eyes.     

Serial analysis of gene expression in the silkworm, Bombyx mori

The silkworm Bombyx mori is one of the most economically important insects and serves as a model for Lepidoptera insects. We used serial analysis of gene expression (SAGE) to derive profiles of expressed genes during the developmental life cycle of the silkworm and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, of which 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool on the EST database, 35% of the tags matched known silkworm expressed sequence tags. SAGE demonstrated that a number of the genes were up- or down-regulated during the four developmental phases of the egg, larva, pupa, and adult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags constituted the most efficient method of gene identification, which facilitated the analysis of a large number of unknown genes.     

Starvation-responsive glycine-rich protein gene in the silkworm Bombyx mori

Four glycine-rich protein (GRP) genes were identified from expressed sequence tags of the maxillary galea of the silkworm. All four genes were expressed in the maxillary pulp, antenna, labrum, and labium, but none of the genes were expressed in most internal organs. Expression of one of the genes, termed bmSIGRP, was further increased approximately fivefold in the mouth region (including the maxilla, antenna, labrum, labium, and mandible) after 24 h of starvation. bmSIGRP expression peaked at 24 h and gradually declined during the subsequent 2 days. When a synthetic diet not containing proteins was fed, bmSIGRP expression increased significantly in the mouth region to levels similar to that observed in starved larvae. Synthetic diets that lacked vitamins or salts but contained amino acids did not significantly affect bmSIGRP expression. These results suggest that amino acid depletion increases bmSIGRP expression.     

Post-translational processing in the synthesis of egg-specific protein in the silkworm,Bombyx mori

The post-translational processing of egg-specific protein (ESP) in developing ovarian follicles of the silkworm, Bombyx mori was analyzed using in vivo and in vitro labeling systems with some radioactive precursors. The labeling with[³⁵S]methionine revealed that ESP is first synthesized as 69 kDa peptide (69K-ESP) which is then converted to 72 kDa peptide (72K-ESP) until 2 h. Some of 72K-ESP molecules were converted to 64 kDa peptide (64K-ESP) after 10h-labeling. [¹⁴C]Mannose was incorporated into 69K-ESP and 72K-ESP. In the presence of tunicamycin, labeling with [³⁵S]methionine brought about a new 67 kDa peptide (67K-ESP) by reducing the incorporation into 69K- and 72K-ESP. [³²P]Ortho-phosphate was incorporated into only 72K-ESP by a 2 h-pulse labeling. Treatment of 72K-ESP with alkaline phosphatase converted it to 69K-ESP. These results along with the available information led to the conclusion that the primary translation product is sequentially processed to 67K-ESP by signal peptide cleavage, to 69K-ESP by glycosylation, and finally to 72K-ESP by phosphorylation as the actual product in the peptide synthesis. The limited conversion of 72K-ESP to 64K-ESP is proposed to be a post-endocytotic event.     

cDNA cloning and mRNA expression of LIM protein gene homologue from the silkworm, Bombyx mori

LIM protein cDNA, from Bombyx mori that contains an open reading frame of 622 bp encoding 94 amino acids, was identified and characterized. The B. mori LIM protein homologue is classified into group 2 LIM proteins that contain glycine-rich LIM domain. B. mori LIM protein mRNA is up-regulated at late embryogenesis and detected in the mid-gut of 5th instar larvae.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori

We designed and constructed a genome-wide microarray with 22,987 70-mer oligonucleotides covering the presently known and predicted genes in the silkworm genome, and surveyed the gene expression in multiple silkworm tissues on day 3 of the fifth instar. Clusters of tissue-prevalent and tissue-specific genes and genes that are differentially expressed in different tissues were identified, and they reflect well major tissue-specific functions on the molecular level. The data presented in this study provide a new resource for annotating the silkworm genome.     

A pattern recognition protein for peptidoglycan. Cloning the cDNA and the gene of the silkworm, Bombyx mori

Peptidoglycan recognition protein (PGRP) specifically binds to peptidoglycan and is considered to be one of the pattern recognition proteins in the innate immunity of insect. The PGRP is an essential component for peptidoglycan to trigger the prophenoloxidase cascade that is now recognized to be an important insect defense mechanism. We cloned cDNA encoding PGRP from the silkworm fat body cDNA library. Northern blot analysis showed that the PGRP gene is constitutively expressed in the fat body, epithelial cell, and hemocytes of naive silkworms. Furthermore, a bacterial challenge intensified the gene expression, with the maximal period being from 6 to 36 h after infection. The upstream sequence of the cloned PGRP gene was shown to contain putative cis-regulatory elements similar to the NF-kappaB-like element, interferon-response half-element, and GATA motif element, which have been found in the promoters of the acute phase protein genes of mammals and insects. A homology search revealed that the homologs of silkworm PGRP are present in mice, nematodes, and bacteriophages. This suggests that the recognition of peptidoglycan as foreign is effected in both vertebrates and invertebrates by PGRP homologs with an evolutionally common origin.     

一个新的家蚕LIMonly蛋白基因的序列分析和原核表达

LIM蛋白是一类含有LIM结构域的真核蛋白家族,它可介导细胞分化和发育,转录调控和激活、参与细胞骨架形成等多种重要的生物调节过程。从家蚕蛹cDNA文库获得一个新的编码LIM only蛋白的基因,我们将其命名为BmLIMO（BombyxmoriLIMonly protein）。该基因ORF长度为561 bp,编码长度为186个氨基酸残基的蛋白质,预测分子量为21.6 kDa,等电点为9.14。对BmLIMO基因进行了生物信息学方面的分析,并用DNA重组技术将Bm-LIMO片段克隆到原核表达载体pET-28α中,在大肠杆菌BL21（DE3）中成功表达了可溶性BmLIMO蛋白,这为进一步研究该蛋白功能奠定了基础。     

Structure and expression analysis of the cecropin-E gene from the silkworm, Bombyx mori

Cecropins belong to the antibacterial peptides family and are induced after injection of bacteria or their cell-wall components. By silkworm cDNA microarray analysis, a novel type of Cecropin family gene was identified as a cDNA up-regulated in early embryo, 1 day after oviposition. The cDNA isolated was 394 bp with 198 ORF translating 65 amino acids, encoding BmCecropin-E (BmCec-E). Using Southern hybridization and genome search analysis, the number of BmCec-E gene was estimated to be at least two per haploid, which consisted of two exons, as in other Cecropin family members. BmCec-E mRNA was expressed transiently 1 day after egg-laying (AEL, germ-band formation stage), and was specifically expressed in the degenerating intestine during the pre-pupal and pupal stages, unlike other Cecropin family genes. Immune challenge analysis showed that BmCec-E gene expression was more strongly induced by Escherichia coli (gram-negative) than by Micrococus luteus (gram-positive), and not by virus injection. By bacterial challenge, expression of BmCec-E mRNA was induced 12 h after injection, and was maintained for 24 h. Expression of BmCec-E after immune challenge was observed strongly in excretory organs, such as hindgut and malphigian, slightly in fat body, skin, and midgut.     

Molecular cloning, expression and characterization of a novel vacuolar protein sorting 4 gene in silkworm, Bombyx mori

The vacuolar protein sorting 4 (Vps4) protein is essential for the multivesicular body (MVB) pathway, virus budding process and cytokinesis. Vps4 has been identified and characterized from many species, but not from silkworm Bombyx mori. In this study, we firstly identified and cloned the silkworm homologous gene for VPS4, expressed it in Escherichia coli, purified and characterized the protein designated as BmVps4. The BmVps4 cDNA contains an open reading frame of 1,314?bp, and encodes a protein of 438 amino acid residues. BmVps4 is of high sequence-similarity to Vps4 proteins from other species. The recombinant BmVps4 shows ATPase activity, which can be stimulated by Mg²⁺ and inhibited by dominant mutations. Together, our data suggest BmVps4 is the genuine silkworm homologue of Vps4. To our knowledge, this is the first-time characterization of any silkworm MVB proteins. This study will facilitate further investigation of silkworm MVB pathway and its possible roles in the infection and budding of B. mori nuclear polyhedrosis virus (BmNPV), which is one of the most common and severe pathogens for silkworms. The cloned BmVps4 sequence is deposited in GenBank (Accession number GQ995504).     

家蚕miRNA Novel-31~*上调溶血素基因的表达

【目的】Novel-31*是在家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus,Bm CPV)感染的家蚕中发现的一个差异表达miRNA。本研究旨在验证Novel-31*对其靶基因表达的调控作用,以便进一步研究miRNA及其靶基因在昆虫免疫调节中的作用。【方法】用生物信息学方法预测Novel-31*的靶基因,荧光定量PCR分析Novel-31*及其靶基因在家蚕感染Bm CPV后不同时间点的表达变化;构建miRNA慢病毒表达载体和靶基因慢病毒表达载体,转染293T细胞,同时合成Novel-31*mimics转染家蚕培养细胞Bm N,使用荧光定量PCR检测Novel-31*对靶基因表达的调控作用。【结果】生物信息学方法预测发现,溶血素基因是Novel-31*的靶基因,其结合位点位于溶血素基因的5'UTR区域。荧光定量PCR分析表明,Novel-31*及溶血素基因在感染Bm CPV的家蚕血淋巴细胞中呈现明显的上调表达。荧光定量PCR检测表明,在Novel-31*慢病毒表达载体和溶血素基因5'UTR慢病毒表达载体转染的293T细胞中和在转染Novel-31*mimics的家蚕Bm N细胞中,溶血素基因都上调表达。【结论】溶血素基因是miRNA Novel-31*的靶基因,Novel-31*与溶血素基因5'UTR结合,上调溶血素基因的表达。     

Sequence structure and expression pattern of a novel anionic defensin-like gene from silkworm (Bombyx mori)

A defensin-like gene, BmdefA, was rediscovered in the silkworm genome and expressed sequence tags databases. The open reading frame of BmdefA encodes a prepropeptide consisting of a 22-residue signal peptide, a 34-residue propeptide, and a 36-residue mature peptide with a molecular mass of 4.0 kDa. The mature peptide possesses the characteristic six-cysteine motif of insect defensins, and its predicted isoelectric point is 4.12, indicating it is a novel anionic defensin. An intron is present in BmdefA and several cis-regulatory elements are in the regulating region. It is transcribed constitutively at a high level in the hemocyte, silk gland, head, and ovary of the silkworm larvae, and in the fat body of early-stage pupae and moth. BmdefA is also strongly induced by immune challenge. These results suggest that BmdefA plays an important role in both immunity and metamorphosis. Hongxiu Wen and Xiqian Lan contributed equally to this work and should be considered co-first authors.     

Transient expression of chimeric CAT genes injected into early embryos of the domesticated silkworm Bombyx mori

In order to establish a transient expression system for genes introduced into early embryos of the silkworm, Bombyx mori, we tested various promoters ligated with CAT reporter genes. The embryos into which we injected supercoiled plasmid DNA of pFb(-860/+10)CAT containing the Bombyx fibroin promoter region ligated to the CAT gene showed a reasonably high CAT activity beginning around 30 h after oviposition. This high activity was observed only when the plasmid was injected before termination of the early nuclear cleavage stage, which was about 8 h after oviposition, but not after this stage. This means that the expression of injected DNA is closely related to the presence of cleavage nuclei in early embryos. Promoters originating from insect genes, like the Bombyx sericin-1 gene, Drosophila hsp70 and Drosophila copia LTR, functioned as strong promoters in the embryos. On the contrary, promoters from mammalian virus genes, such as the SV40 early and Moloney murine leukemia virus LTR genes, functioned as weak promoters. Moreover, linearized DNAs showed no or weak activity of expression in embryos. From these results, we conclude that the silkworm embryo transient expression system is a useful tool for studying the mechanism of regulation of insect genes.