Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

Studies on silk fibroin of the silkworm Bombyx mori

A procedure has been developed to obtain native fibroin in a pure state from the reservoir part of the silk gland. The purified protein has a sedimentation coefficient of 10 S as determined on sucrose density gradients and the amino acid composition is similar to that reported for fibroin from the cocoons. The effects of various solvents has been studied; lithium thiocyanate was found to be the solvent of choice. By in vivo labeling of fibroin with [³H]glycine and [¹⁴C]alanine it was demonstrated that fibroin synthesized in the posterior part of the gland and that stored in the reservoir part are identical.     

Sperm-mediated gene transfer in the silkworm Bombyx mori

Plasmid DNA containing the CAT reporter gene was injected into the testis of V instar silkworm larvae. The persistence, expression, and transmission of the injected DNA were monitored in the injected individuals till eclosion as well as in the progeny. The DNA injected into the testis persisted extrachromosomally during the entire period of metamorphosis and was also transferred into the egg via sperm during fertilization. Injected plasmids were rescued from the moths that emerged from the injected larvae and also from the eggs laid by the moths that copulated with injected males. Positive signals for CAT assay in the experimental samples suggested that the injected DNA was internalized in the testicular cells and sperm. The persistence, expression, and transmission of the DNA injected into the testes indicate that sperm-mediated gene transfer is possible in the silkworm, Bombyx mori. Arch. Insect Biochem. Physiol. 37:168–177, 1998. © 1998 Wiley-Liss, Inc.     

Sequence polymorphisms in the 5'-upstream region of the fibroin H-chain gene in the silkworm, Bombyx mori

DNA fragments containing the fibroin H-chain gene from two different strains of Bombyx mori, J-139 and Nd (2) were cloned into phage lambda Charon 4A. Comparison of the restriction sites in these cloned DNAs revealed that in addition to the known polymorphism in the region coding for the repetitive amino acid sequence of the fibroin H-chain [Manning and Gage, J. Biol. Chem. 255 (1980) 9451-9457], at least two other types of polymorphism were present, one around the 5' end of the structural gene, and the other in the far upstream region of the gene. Restriction sites around the 5' end of the gene were well conserved between these strains, but some heterogeneity, suggesting the presence of small insertions, deletions or base changes, was noted. In contrast, DNA sequences of the region 2-4 kb upstream from the 5' end of the gene were markedly different between these two strains, indicating that either a deletion or an insertion of a DNA sequence longer than 2 kb had occurred in this region. Comparison with several other strains suggested that the observed changes in the far-upstream region were unique to the Nd(2) strain.     

pH induced changes in the rheology of silk fibroin solution from the middle division of Bombyx mori silkworm

The rheological properties of fibroin silk solutions extracted from the middle division of Bombyx mori silkworms were examined. Acidification of the solutions with acetic acid vapor gelled the material, a process which at short time scales could be reversed by exposure to ammonia vapor. The solution could also be converted to sol from the gel state by the addition of EDTA. The possible mechanisms for gel formation in fibroin solutions is discussed as are the implications for the process of spinning silk fibers.     

Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection

Targeted mutagenesis is one of the key methods for functional gene analysis. A simplified variant of gene targeting uses direct microinjection of custom-designed Zinc Finger Nuclease (ZFN) mRNAs into Drosophila embryos. To evaluate the applicability of this method to gene targeting in another insect, we mutagenized the Bombyx mori epidermal color marker gene BmBLOS2, which controls the formation of uric acid granules in the larval epidermis. Our results revealed that ZFN mRNA injection is effective to induce somatic, as well as germline, mutations in a targeted gene by non-homologous end joining (NHEJ). The ZFN-induced NHEJ mutations lack end-filling and blunt ligation products, and include mainly 7 bp or longer deletions, as well as single nucleotide insertions. These observations suggest that the B. mori double-strand break repair system relies on microhomologies rather than on a canonical ligase IV-dependent mechanism. The frequency of germline mutants in G₁ was sufficient to be used for gene targeting relying on a screen based solely on molecular methods.     

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.     

家蚕丝素P25蛋白基因启动子顺式作用元件的功能分析

为了阐明蚕丝蛋白基因表达调控的分子机制, 利用Bac-to-Bac杆状病毒表达系统及实时荧光定量PCR等技术, 对家蚕Bombyx mori丝素P25基因启动子上游1 233 bp的活性及其调控元件的功能进行了分析。结果表明： 在P25启动子上游-423~-1 233区和-127~-238区存在正调控元件, 在-238~-423区段存在负调控元件; PSGF和BMFA两结合元件在P25基因表达中起负调控作用。PSGF结合元件对A3启动子在后部丝腺的活性具有一定的增强作用, 进一步验证了PSGF调控元件的功能。通过对P25基因启动子的活性分析, 尤其是对PSGF和BMFA调控元件的功能分析, 有利于进一步了解P25基因表达调控的精细机制。     

Structural analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori

To study the minimal length required for the secretion of recombinant proteins and silkproteins in posterior silk gland,the signal peptide(SP)of the fibroin heavy chain(FibH)of silkwormBombyx mori was systematically shortened from the C-terminal.Its effect on the secretion of protein wasobserved using enhanced green fluorescent protein(EGFP)as a reporter.Secretion of EGFP fusion proteinswas examined under fluorescence microscope.FibH SPs with lengths of 20,18,16 and 12 a.a.can directthe secretion of the reporter,yet those with lengths of 11, 10, 9, 8 and 1 a.a. can not. When the FibH SP wasshortened to 12a.a., the secretion efficiency was decreased slightly and cleavage occurred within EGFP.When 16a.a.of the FibH SP were used,the secretion of fusion protein was normal and the cleavage sitewas between the Gly-Ser linker and Met,the starting amino acid of EGFP. These findings are applicable forthe expression of foreign proteins in silkworm silk gland.The cleavage site of the SP is discussed andcompared with the predictive results of the SignalP 3.0 online prediction program.     

家蚕卵黄原蛋白及其受体基因

家蚕小卵突变体 (Smalleggmutant ,sm) ,其卵体积仅及正常卵的 2 / 3,不能受精而致死。因其卵母细胞不能正常吸收卵黄原蛋白 (Vg) ,人们认为其原因可能是卵黄原蛋白受体基因 (VgR)突变所致。本研究首先通过克隆筛选和基因组序列分析 ,获得了 2 5 6 4bp含有 ployA的家蚕卵黄原蛋白受体基因 (BmVgR)片段。将该基因片段的预测蛋白与其它物种的VgR/YPR和低密度脂蛋白受体 (LDLR)家族比较 ,发现该基因具有LDLR家族的基本结构特征。其次 ,经RT PCR检测 ,结果表明BmVgR在sm的不同时期的卵母细胞中都能正常转录。最后 ,分别对sm不同发育时期的体液和卵的总蛋白进行SDS PAGE分析 ,发现该突变体的卵母细胞不能正常摄取体液蛋白 (包括Vg)。综合分析 ,sm不能正常摄取Vg ,可能并不是VgR的功能异常导致 ,而是与滤泡细胞异常有关     

Cloning and characterization of nanos gene in silkworm Bombyx mori

Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5＇-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mor/and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5.38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee （Apis mellifera）. This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.     

Fine organization of Bombyx mori fibroin heavy chain gene

The complete sequence of the Bombyx mori fibroin gene has been determined by means of combining a shotgun sequencing strategy with physical map-based sequencing procedures. It consists of two exons (67 and 15 750 bp, respectively) and one intron (971 bp). The fibroin coding sequence presents a spectacular organization, with a highly repetitive and G-rich (~45%) core flanked by non-repetitive 5′ and 3′ ends. This repetitive core is composed of alternate arrays of 12 repetitive and 11 amorphous domains. The sequences of the amorphous domains are evolutionarily conserved and the repetitive domains differ from each other in length by a variety of tandem repeats of subdomains of ~208 bp which are reminiscent of the repetitive nucleosome organization. A typical composition of a subdomain is a cluster of repetitive units, Ua, followed by a cluster of units, Ub, (with a Ua:Ub ratio of 2:1) flanked by conserved boundary elements at the 3′ end. Moreover some repeats are also perfectly conserved at the peptide level indicating that the evolutionary pressure is not identical along the sequence. A tentative model for the constitution and evolution of this unusual gene is discussed.     

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.     

Expression analysis of decapentaplegic gene in the silkworm,Bombyx mori

The decapentaplegic (dpp) gene in Drosophila is involved in multiple developmental processes, and is a highly conserved among various eukaryotic species, including Bombyx mori. Although the gene has well been characterized in Drosophila species the B. mori dpp has not yet been functionally analyzed. In this study, we analyzed the expression pattern of B. mori dpp in 12 different developmental days/stages (7 days for fifth instar larvae, 2 days for spinning stage, 2 days for pupal stages, and 1 day for adults) in both male and female silkworms using quantitative real‐time RT‐PCR (qRT‐PCR). mRNA expression of B. mori dpp was much higher in the female larvae up to the mid‐stage of the fifth instar compared with the corresponding male larvae. Similarly, dpp expression also was much higher in females during the eclosion period than that in the corresponding male pupae. During the embryonic stage, the expression level of the dpp gene was much higher compared to that of adult stage in both male and female silkworms. These results suggest that the B. mori dpp gene plays multiple roles in the developmental of B. mori.     

Characterization of a gene encoding prohibitin in silkworm, Bombyx mori

Background

Prohibitin (PHB) is an evolutionarily conserved multifunctional protein with ubiquitous expression. However, its molecular roles are largely unknown.

Methods

To better understand the function of prohibitin protein in silkworm (BmPHB), its coding sequence was isolated from a cDNA library of silkworm pupae. An His-tagged BmPHB fusion protein was expressed in Escherichia coli Rosetta (DE3) and purified with affinity and reversed-phase chromatography. Purified rBmPHB was used to generate anti-BmPHB polyclonal antibody. The subcellular localization of BmPHB was analysed by immunohistochemistry.

Results

BmPHB gene has an ORF of 825 bp, encoding a predicted peptide with 274 amino acid residues. Immunostaining indicate that prohibitin is expressed in nucleus and predominately in cytoplasm. Western blot analyses indicated that, in the fifth instar larva, BmPHB was expressed descendingly in gonad, malpighian tubule, trachea, fatty body, intestine, and head. However, no expression was detected in larva's silk gland and epidermis. In addition, BmPHB was expressed in the nascent egg, larva and pupa, but not in the moth.

Conclusions

The expression of BmPHB gene presents differential characteristic in different stage and tissues. It may play important roles in the development of silkworm.

General significance

Studies on prohibitin have been still restricted to a few specific insects and insect cell lines such as Drosophila, Acyrthosiphon pisum and mosquito cell lines, not yet in silkworm. This is a first characterization of prohibitin in silkworm, B. mori.