Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

Characterization and purification of the biologically active v-sis protein generated in silkworm larvae with the Bombyx mori nuclear polyhedrosis virus vector system

The v-sis oncogene of simian sarcoma virus encodes a protein which is homologous to the human platelet-derived growth factor B-chain. The biologically active v-sis protein was expressed in silkworm larvae using the Bombyx mori nuclear polyhedrosis virus vector system. The v-sis protein purified from infected silkworm larvae is a 30 kDa disulfide-linked homodimer. Mitogenic activity of the v-sis protein was comparable to that of PDGF and inhibited by the pretreatment with anti-PDGF antibody. These results show that the recombinant v-sis protein is biologically and antigenically related to PDGF.     

X-ray structural study of noncrystalline regenerated Bombyx mori silk fibroin

X-ray diffraction measurements of regenerated Bombyx mori silk fibroin were carried out to determine its structural characteristic from an analysis of differential radial distribution functions (DRDFs). The temperature dependence of X-ray diffraction patterns from noncrystalline and crystal structures of regenerated silk fibroin was investigated using a high temperature furnace. Time resolved X-ray diffraction profiles were also obtained to construct kinematical models of structural changes caused by the addition of water. DRDFs, calculated from the experimental data, were compared with the DRDFs simulated on the basis of the Monte Carlo method. In order to model the noncrystalline structures, structural units were assumed to be parts of the crystalline structure of silk and those with appropriate structural defects reported previously. From the comparison of experimental and simulated DRDFs, it was determined that noncrystalline regenerated silk consisted of locally ordered atomic sheets similar to the atomic arrangement in the silk I crystal (Type-I sheets), and the final state of the structural change was noncrystalline, consisting of small crystallites, the structure of which is similar to that of silk II (Type-II crystallites). Time resolved DRDFs were also qualitatively interpreted by both the ordering of Type-I sheets and structural changes from Type-I to Type-II. The formation of the small Type-II crystallites obtained in this study was consistent with the nucleation of silk II by birefringence measurements of silk glands and the spinneret of Bombyx mori silkworm reported previously. X-ray diffraction should be a useful technique to understand the structural characteristics of noncrystalline organic materials.     

Laser-Raman spectroscopic studies of the eggshell (chorion) of Bombyx mori

Laser-Raman spectroscopic studies of the eggshell (chorion) of the silkmoth Bombyx mori reveal that its component proteins consist of 60–70% antiparallel β-pleated sheet and 30–40% of β-turns. The disulphide bonds, which crosslink the (extremely rich in cysteine)-proteins of the outer lamellar eggshell layer, are apparently found in G-G-G (gauche-gauche-gauche) and T-G-T (trans-gauche-trans) conformation; there is no evidence for the existence of free sulphydryls. The highly localized tyrosine residues appear to form hydrogen bonds, acting as weak proton donors or as acceptors.     

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.     

A genetic diversity study of silkworm using cleaved amplified polymorphic sequence (CAPS) markers

The silkworm, Bombyx mori is a beneficial insect of great economic importance in China for its silk production. In this study, we obtained 11 cleaved amplified polymorphic sequence (CAPS) markers and one PCR polymorphism marker from the genes of the silkworm, B. mori. A backcross progeny analysis showed that all these molecular markers were segregated in a Mendelian fashion and that polymorphisms were co-dominant. These markers were used to investigate the genetic diversity among 29 strains of B. mori from China, Japan and Europe. Cluster analysis, based on the genetic similarities calculated from CAPS data, grouped these strains roughly according to their geographical origin. One group contained silkworm strains from Europe and some of the Japanese strains were interspersed into the Chinese groups, whereas other Japanese strains clustered together.     

Differential effects of sugar-mimic alkaloids in mulberry latex on sugar metabolism and disaccharidases of Eri and domesticated silkworms: enzymatic adaptation of Bombyx mori to mulberry defense

Mulberry leaves (Morus spp.) exude latex rich in sugar-mimic alkaloids, 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1) and 1-deoxynojirimycin (DNJ), as a defense against herbivorous insects. Sugar-mimic alkaloids are inhibitors of sugar-metabolizing enzymes, and are toxic to the Eri silkworm, Samia ricini, a generalist herbivore, but not at all to the domesticated silkworm, Bombyx mori, a mulberry specialist. To address the phenomena, we fed both larvae diets containing different sugar sources (sucrose, glucose or none) with or without sugar-mimic alkaloids from mulberry latex. In S. ricini, addition of sugar-mimic alkaloids to the sucrose (the major sugar in mulberry leaves) diet reduced both growth and the absorption ratio of sugar, but it reduced neither in B. mori. The midgut soluble sucrase activity of S. ricini was low and inhibited by very low concentrations of sugar-mimic alkaloids (IC₅₀=0.9–8.2 μM), but that of B. mori was high and not inhibited even by very high concentrations (IC₅₀>1000 μM) of sugar-mimic alkaloids. In S. ricini, the addition of sugar-mimic alkaloids to the glucose diet still had considerable negative effects on growth, although it did not reduce the absorption ratio of glucose. The hemolymph of S. ricini fed sugar-mimic alkaloids contained sugar-mimic alkaloids. The trehalose concentration in the hemolymph increased significantly in S. ricini fed sugar-mimic alkaloids, but not in B. mori. The trehalase activities of S. ricini were lower and inhibited by lower concentrations of sugar-mimic alkaloids than those of B. mori. These results suggest that sugar-mimic alkaloids in mulberry latex exert toxicity to S. ricini larvae first by inhibiting midgut sucrase and digestion of sucrose, and secondly, after being absorbed into hemolymph, by inhibiting trehalase and utilization of trehalose, the major blood sugar. Further, our results reveal that B. mori larvae evolved enzymatic adaptation to mulberry defense by developing sucrase and trehalase that are insensitive to sugar-mimic alkaloids.     

Induction of antibacterial protein synthesis by soluble peptidoglycan in isolated fat body from larvae of the silkworm, Bombyx mori

Synthesis and secretion of bactericidal protein (cecropin) and lysozyme were induced by soluble peptidoglycan fragments (SPG) from Escherichia coli in a culture of fat body from Bombyx mori larvae. The rate of the secretion by fat body increased as a function of SPG concentration added to the culture medium. The induction of bactericidal activity was specific for peptidoglycan of a particular structure. Thus, SPG from Micrococcus luteus was 500-times less potent than E. coli SPG, and various glucans and peptides structurally related to peptidoglycan were all ineffective as elicitor. These results support the hypothesis that bacteria invading the haemocoel have to be partially degraded to generate peptidoglycan fragments as a signal molecule, which subsequently acts on a receptor on fat body cells and induces antibacterial protein synthesis.     

A target protease activity of serpins in insect hemolymph

Two zymogens of the serine enzymes (prophenoloxidase activating enzyme and BAEEase, an enzyme hydrolyzing ethyl ester), which are thought to be components of prophenoloxidase cascade in silkworm (Bombyx mori) plasma, were activated through the action of microbial cell wall components. The two active enzymes of the zymogens were studied with regard to the regulation of their activities by two endogenous serpins (silkworm anti-trypsin and silkworm anti-chymotrypsin).

BAEEase activity was shown to be inactivated by silkworm antitrypsin, whereas the inactivation of prophenoloxidase activating enzyme by either of silkworm antitrypsin and silkworm antichymotrypsin could not be demonstrated under the experimental conditions.     

Solution structure of paralytic peptide of silkworm,Bombyx mori

Paralytic peptide of Bombyx mori (BmPP) is one of the multifunctional ENF-peptides; the name of “ENF” is the consensus N-terminal amino acid sequence of the family peptides. We revealed that BmPP significantly possesses growth-blocking activity and plasmatocyte-spreading activity and that its activity profiles are different from those of another ENF-family peptide, namely, the growth-blocking peptide of Pseudaletia separata (PsGBP). We also determined the NMR structures of BmPP and PsGBP under the same conditions, which revealed the structural differences of the first and second β-turn regions between the two peptides. On the basis of our results, it can be considered that the tertiary structural difference in these peptides may cause their different profiles of growth-blocking activity.     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, ¹³C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol–gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were -helix and random coil in solution. There also existed a little β-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no -helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol–gel transformation. The velocity increased obviously when the temperature was above 30 °C. During the sol–gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to β-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.     

Uptake of the sex pheromone H-bombykol and related compounds by male and female Bombyx antennae

The uptake of ³H-bombykol and related compounds was investigated on the antennae of the silkmoth Bombyx mori with elution and liquid scintillation techniques. The pheromone uptake is a sum of several time-dependent processes and more or less susceptible against pretreatment with organic solvents, buffers and SH-inhibitors. Before and after enzymatic conversion the pheromone molecules are mainly adsorbed non-covalently (99 per cent) onto micromolecular structures.     

Distribution of cuticular protein mRNAs in silk moth integument and imaginal discs

The distributions of mRNAs for two cuticular proteins of Hyalophora cecropia were examined with RT-PCR and in situ hybridization. For major regions of larval and pupal cuticle, there was a strong correspondence between the type of cuticle and the predominant cuticular protein message found. Epidermal cells underlying soft cuticle had mRNA for HCCP12, with a RR-1 consensus attributed to soft cuticle, while the epidermal cells associated with hard cuticle had predominantly mRNA for HCCP66, a protein with the RR-2 consensus attributed to hard cuticle. Both messages were found in all areas of the pupal fore- and hind-wings, with modest area-specific difference in concentration being much less than differences in the relative abundance of these cuticular proteins.

mRNA for HCCP12 was present in imaginal discs of feeding larvae of H cecropia. Data from Bombyx mori available at SilkBase (http://www.ab.a.u-tokyo.ac.jp/silkbase/) revealed that imaginal discs from feeding larvae had abundant mRNA for RR-1 cuticular proteins, representing six distinct gene products. Only discs from spinning larvae had mRNAs that coded for RR-2 proteins arising from 10 distinct genes. Thus, lepidopteran wing imaginal discs can no longer be regarded as inactive in larval cuticle production.     

家蚕幼虫BmToll9基因对肽聚糖和金黄色葡萄球菌的响应表达

【目的】Toll信号通路是昆虫中重要的免疫信号通路,其中Toll受体在保持Toll通路的正常免疫应答、抵抗外源病原体中起到关键的作用。本研究旨在探究肽聚糖和金黄色葡萄球菌Staphylococcus aureus对家蚕Bombyx mori Toll受体基因BmToll9-1和BmToll9-2表达的影响。【方法】将革兰氏阳性菌细胞壁主要成分肽聚糖和革兰氏阳性菌金黄色葡萄球菌分别注射感染家蚕5龄第1天幼虫,诱导其发生免疫反应;采用实时荧光定量PCR分析注射后不同感染时间点BmToll9-2和BmToll9-1基因在家蚕幼虫中肠、表皮、脂肪体和丝腺中的相对表达水平。【结果】往家蚕5龄幼虫中注射肽聚糖或金黄色葡萄球菌后,BmToll9-2基因出现了时间和组织的差异性表达。注射肽聚糖和金黄色葡萄球菌均能诱导5龄幼虫中肠BmToll9-2基因的表达上调,注射肽聚糖和金黄色葡萄球菌分别在3和6 h时对基因表达的诱导效果最好,且注射金黄色葡萄球菌比注射肽聚糖对基因表达的诱导效果更好。注射金黄色葡萄球菌能引起5龄幼虫表皮、脂肪体和丝腺中BmToll9-2基因的表达上调,分别于注射后24, 6和24 h时诱导效果最好。注射金黄色葡萄球菌亦能诱导同源的BmToll9-1基因的上调表达。【结论】家蚕幼虫BmToll9基因在肽聚糖或金黄色葡萄球菌注射处理后均能在不同组织中发生上调表达,推测BmToll9基因参与了家蚕对肽聚糖和金黄色葡萄球菌的免疫反应。     

Purification and partial amino acid sequence of initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori

We have purified initiatorin, a prostatic endopeptidase that initiates the protein-arginine degradation cascade in the spermatophore of Bombyx mori. Purification of the enzyme from spermatophores was monitored by measuring BAEE (N-benzoyl- -arginine-ethyl ester) hydrolyzing activity. Spermatophores were used as a source for this enzyme. Of several isoforms the major form (MW, 29 kDa) was purified over 200-fold. The N-terminal sequence of initiatorin showed strong homology with those of serine-type of endopeptidases.     

CRISPR/Cas9系统在昆虫中的应用

CRISPR/Cas9(clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9)技术是一种RNA引导的基因组靶向编辑技术,能对基因组序列进行精确编辑,在探究基因功能、修复受损基因、沉默有害基因、改良品质性状等方面具有广阔的应用前景。近年来,随着对CRISPR/Cas9系统研究的不断深入和改造,该系统以其操作简易、省时、高效等优点在生物学研究的众多领域中得以推广和应用,特别是在果蝇(Bombyx mori)、家蚕(silkworm)、埃及伊蚊(Aedes aegypti)和蝴蝶(butterfly)等多种昆虫中。本文概述了CRISPR/Cas9的结构、作用原理及发展优化,总结了CRISPR/Cas9导入昆虫的策略和在昆虫中的应用,以及对CRISPR/Cas9系统产生脱靶问题的应对策略,以期对经济昆虫和有益昆虫的分子育种、害虫的生物技术防控等研究提供参考。