The genome sequence of silkworm, Bombyx mori.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.     

Serine hydroxymethyltransferase from the silkworm Bombyx mori: Identification,distribution, and biochemical characterization

Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of serine and tetrahydrofolate (THF) to glycine and methylenetetrahydrofolate. cDNA encoding Bombyx mori SHMT (bmSHMT) was cloned and sequenced. The deduced amino acid sequence consisted of 465 amino acids and was found to share homology with other SHMTs. Recombinant bmSHMT was overexpressed in Escherichia coli and purified to homogeneity. The enzyme showed optimum activity at pH 3.0 and 30°C and was stable under acidic conditions. The K_m and k_cat/K_m values for THF in the presence of Nicotinamide adenine dinucleotide phosphate (NADP⁺) were 0.055 mM and 0.081 mM^?1 s^?1, respectively, whereas those toward NADP⁺ were 0.16 mM and 0.018 mM^?1 s^?1 and toward l ‐serine were 1.8 mM and 0.0022 mM^?1 s^?1, respectively. Mutagenesis experiments revealed that His119, His132, and His135 are important for enzymatic activity. Our results provide insight into the roles and regulation mechanism of one‐carbon metabolism in the silkworm B. mori.     

Proteome profile of spinneret from the silkworm,Bombyx mori

The silkworm spinneret is an important tissue for silk fibrillogenesis and spinning. All biochemical processes during silk fibrillogenesis are correlated with silk properties. Understanding the role of spinneret in silk fibrillogenesis may help to reveal the mechanism of silk fibrillogenesis as well as improve silk quality for commercial purposes. Thus, we profiled the proteome of silkworm spinneret. A total of 1572 proteins and 232 differential abundance proteins were identified. Silk fibrillogenesis‐related proteins, such as cuticle proteins, ion‐transporting proteins, muscular proteins, and energy metabolic proteins, were abundant in spinneret. Metabolic pathway and GO enrichment analyses revealed that the identified proteins were involved in energy metabolism, chitin binding, and cuticle construction. Active energy metabolism may provide abundant energy for the muscle contraction as well as ion and water exchange. The chitin binding and cuticle construction process may provide sufficient shear forces for silk formation. Our data suggest that silkworm spinneret provides a suitable physiological and biochemical environment for silk fibrillogenesis. These proteins are potential targets for improving silk quality in the silk industry. Data are available via ProteomeXchange with identifier PXD004455.     

家蚕para钠通道cDNA片段克隆与序列分析

昆虫神经系统para型钠离子通道是拟除虫菊酯类杀虫剂的主要靶标,已有的研究表明钠离子通道基因发生点突变与昆虫对菊酯类杀虫剂的抗性密切相关。本文通过RT-PCR方法克隆获得了编码家蚕Bombyx mori L.钠离子通道的cDNA片段(GenBank No.EF521818),该片段全长4882bp,部分ORF包含3986bp核苷酸,翻译成1328个氨基酸。蛋白序列分析表明,PCR扩增获得的家蚕钠离子通道cDNA片段所编码的氨基酸与其他昆虫的para型钠离子通道α亚基的氨基酸具有很高的同源相似性,与棉铃虫Heliothis virescens Fabricius、埃及伊蚊Aedes aegypti L.、德国小蠊Blattella germanica L.、果蝇Drosophila melanogaster Meigen和家蝇Musca domestica L.的相似性分别为95%、82%、80%、79%、77%。     

家蚕Aly/REF的基因克隆、序列分析及其细胞定位

RNA和输出因子结合蛋白（RNA and export factor binding proteins, REF/Aly）参与RNA的稳定、加工和输出。本研究参照已公开的家蚕Bombyx mori aly序列（GenBank登录号： DQ497195.1）,通过RT-PCR克隆了家蚕aly/ref基因（Bmaly/ref）。测序结果显示,该基因的开放读码框为765 bp,编码254个氨基酸残基, BmAly/REF与黑腹果蝇Drosophila melanogaster、小鼠Mus musculus的同源体的氨基酸序列一致性分别为49.7%和52.7%。结构预测结果显示,BmAly/REF具有REF家族的与RNA结合的结构域RRM,N和C端分别具有REF-N和REF-基序。进化分析结果显示,昆虫的ALY/REF聚为一类,BmAly/REF与赤拟谷盗Tribolium castaneum、意大利蜜蜂Apis mellifera的Aly/REF较为接近。将Bmaly/ref基因克隆进pGS21a(+)载体进行原核表达,重组蛋白免疫小鼠,获得鼠抗BmAly/REF多抗。免疫荧光实验结果显示,BmAly/REF在细胞质和细胞核中均有分布,但主要分布在细胞核中。芯片数据分析显示Bmaly/ref基因在家蚕幼虫5龄第3天各组织中均有较高水平的表达。研究结果显示BmAly/REF可能在RNA的核输出方面发挥作用,为进一步探讨BmAly/REF的功能奠定了基础。     

Isolation and nucleotide sequence of cecropin B cDNA clones from the silkworm, Bombyx mori.

Two cDNA clones encoding cecropin B, an antibacterial protein, were isolated from a fat body cDNA library of the silkworm, Bombyx mori. Amino acid sequences of these clones, deduced from nucleotide sequences, were identical, including signal peptide regions. However, the nucleotide sequences were different at 30 positions. Deduced amino acid sequences of Bombyx mori cecropin B showed higher homology with cecropins from Lepidoptera than with those from Diptera.     

Isolation of the diapause hormone from the silkworm,Bombyx mori

The diapause hormone (DH) secreted from the suboesophageal ganglion is responsible for the embryonic diapause in the silkworm, Bombyx mori. It was extracted from two million mated male adult heads. At least two species of DH were separated from the extracts (a complex lipid fraction) by gel permeation chromatography with Sephadex LH-20 by organic eluants. One of them was further purified by repeating gel permeation column chromatography with Merckogel^® Type OR 6000 in the cold and dark to give finally a single peak with high DH activity. This preparation seems to be chemically pure, and its molecular weight is chromatographically estimated to be between 2000 and 4000. Other characteristics of DH are also presented. Biological activity of the final DH preparation is discussed with reference to other insect hormones such as juvenile hormone and α-ecdysone.     

Storage proteins in the silkworm,Bombyx mori

In the silkworm, Bombyx mori, two storage proteins named SP-1 and SP-2 were shown to decline in concentration in the haemolymph and increase in the fat body during the larval-pupal transformation, when protein granules are formed in the fat body at the same time as the degeneration of mitochondria and endoplasmic reticulum. At the larval-pupal ecdysis, in females the two proteins account for 60% of total fat body protein (80% of the soluble protein), while males have very little SP-1 and SP-2 comprises only 20% of the total fat body protein. The concentration of protein granules in the fat body cytoplasm is much greater in females than in males, and the granules in females have partially crystalline inner zones. This is different from males where granules with non-crystalline structure are most numerous.The properties of these proteins purified from pupal fat body are similar to those of Cecropia storage proteins and calliphorin, all of which have molecular weights of around 500,000 and are composed of subunits of mol. wt about 85,000. SP-1 differs from SP-2 by having an exceptionally high content of methionine, but much less glutamate, phenylalanine and tyrosine. SP-1 resembles another female-specific protein, vitellogenin and SP-2 resembles calliphorin in amino acid composition.From these results, it is concluded that SP-1 and SP-2 have storage roles and are deposited in protein granules.     

Chymotrypsin inhibitors from hemolymph of the silkworm, Bombyx mori.

Three new protease inhibitors were isolated and purified about 200-fold from hemolymph of silkworm larvae, Bombyx mori, using ion-exchange and affinity chromatography. Two of the three inhibitors were basic proteins (SCI-I had pI 9.4 and SCI-II had pI 9.6) and one was acidic (SCI-III had pI 4.0). The molecular weight of each inhibitor was determined to be 7,000 by the sedimentation equilibrium method. The amino acid composition of the inhibitors were similar except for the contents of Asp, Glu, Ile, Leu, and Lys. Val, His, and Trp were not present in the inhibitors and Met appeared only in SCI-III. The CD spectra of the inhibitors were all similar and indicated a low content of alpha-helical structure (10% at most). Each inhibitor could inhibit the protease and esterase activities of bovine alpha-chymotrypsin at a one-to-one molar ratio, and the dissociation constants were 3.1 X 10(-9)M for SCI-I and II and 1.3 X 10(-8)M for SCI-III. Only SCI-II showed a weak inhibitory activity against bovine trypsin. Subtilisin BPN' and papain were not inhibited by these inhibitors.     

一种家蚕类浓核病毒的组织病理学特征

本文从家蚕病蚕中分离到一种家蚕类浓核病毒（BmDNV-Like）,对它的组织病理学研究表明:该病毒首先寄生家蚕中肠柱状细胞,继而引起其细胞核的膨大和破裂;组织原位杂交结果表明该病毒既能在家蚕中肠柱状细胞中增殖,也能在中肠的杯形细胞中增殖,甚至在感染后期能在家蚕幼虫的大部分组织细胞中感染和增殖。     

Purification and molecular properties of vitellin from the silkworm,Bombyx mori

Vitellin was purified from the eggs of the silkworm, Bombyx mori by a simple method which included a specific precipitation at pH 6 under low ionic concentration and DEAE-cellulose column chromatography. The final preparation was highly homogeneous as judged by gel electrophoresis, electron microscopy and ultracentrifugation.Vitellin was defined as glycolipoprotein with a sedimentation coefficient (S_{20, W}) of 13.5S and a molecular weight of 440,000. The molecule was almost spherical in shape with a diameter of 13 nm. The molecule contained 3% mannose and 7.5% total lipids which comprised triacylglycerol, diacylglycerol, cholesterol, phosphatidylcholine and phosphatidylethanolamine. The amino acid composition displayed a high content of glutamic and aspartic acids and a low content of methionine. The molecule was composed of two non-identical subunits with molecular weights of 180,000 and 42,000, and the native molecule was assumed to be a tetramer composed of two molecules of each of these subunits. Separation of the two subunits was achieved, and mannose was covalently associated only with the heavier subunit.The rabbit anti-egg vitellin antibody cross-reacted with the haemolymph vitellogenin but not with other haemolymph proteins, nor with the vitellogenin from Locusta migratoria. The antibody also reacted with the haemolymph vitellogenin of the silkworm, Philosamia cynthia.     

Purification and characterization of cocoonase from the silkworm Bombyx mori

Cocoonase (CCN) which facilitates the degradation of a cocoon is recognized as a trypsin-like serine protease. In this study, CCN from the silkworm Bombyx mori was purified and comprehensively characterized. Its activity was maximal at about pH 9.8. It was stable above pH 3.4 at 4?°C and below 50?°C at pH 7.5. CuSO₄, FeSO₄, and ZnSO₄ showed inhibitory effects on CCN, but other salts improved activity. Typical trypsin inhibitors inhibited CCN, but the relative inhibitory activities were much lower than those against bovine trypsin. An extract of cocoon shells inhibited trypsin, but it was only slightly inhibitory against CCN. There were significant differences in catalytic efficiencies and substrate specificities as between CCN and bovine trypsin.