Molecular cloning of the prothoracicotropic hormone from the tobacco hornworm,Manduca sexta

A cDNA encoding a putative precursor of prothoracicotropic hormone (PTTH) from the tobacco hornworm, Manduca sexta, was isolated and sequenced. This clone contains an open reading frame encoding a 226-amino acid prepropeptide hormone. The deduced amino acid sequence is composed of a signal sequence, a precursor domain and a mature hormone and shows similarities to the other PTTHs that have been cloned from closely related lepidopteran species, Bombyx mori, Samia cynthia ricini, Antheraea peryni, and Hyalophora cecropia. Although these cDNAs showed slightly less similarities in predicted amino acid sequences, seven cysteine residues and the hydrophobic regions within those mature peptides were conserved. In situ hybridization using a cDNA probe encoding the Manduca PTTH showed that PTTH mRNA was in two pairs of neurosecretory cells in the Manduca brain. The recombinant putative Manduca PTTH produced in E. coli was biologically active, both causing a larval molt in neck-ligated Manduca 4th instar larvae (ED(50)=50 pM) and the adult molt of diapausing Manduca pupae (ED(50)=79 pM), but was unable to stimulate molting of debrained Bombyx pupae.     

Distinctive presence of peritracheal athrocytes in Bombyx mori L. and Bombyx mandarina M. as compared to their absence in several other lepidopteran species

Pericardial cells are present in a wide variety of insects and are thought to constitute the majority of 'athrocytes (nephrocytes)'. In contrast, peritracheal athrocytes have only been observed in Bombyx mori L. Although peritracheal athrocytes have a distinct morphology, it is unknown whether these cells are common to all lepidopterans. We anatomically compared eight lepidopteran species: Bombyx mori L. and Bombyx mandarina M. (Bombycidae); Samia cynthia ricini D. (Saturniidae); Agrius convolvuli L. (Sphingidae); Spodoptera litura F. and Mythimna separata W. (Noctuidae); Pieris rapae L. (Pieridae); and Glyphodes pyloalis W. (Crambidae). Of these species, only Bombyx mori L. and Bombyx mandarina M. possess peritracheal athrocytes.     

Prothoracicotropic activity of SBRPs, the insulin-like peptides of the saturniid silkworm Samia cynthia ricini

Synthesis and secretion of the insect molting hormone ecdysteroid in the prothoracic glands (PGs) are stimulated by the prothoracicotropic hormone (PTTH) secreted by the brain. Bombyxins, insulin-like peptides of the silkworm Bombyx mori, show prothoracicotropic activity when administered to the saturniid silkworm Samia cynthia ricini, but they are inactive to B. mori itself. Recently, the genes for the bombyxin homologs of S. cynthia ricini (referred to as Samia bombyxin-related peptides, SBRPs) were cloned. To examine the prothoracicotropic activity of SBRPs on S. cynthia ricini, we synthesized two representative molecules, SBRP-A1 and -B1. They promoted pupa-to-adult development with ED(50) of 50 and 10 ng/pupa (EC(50) of 5 and 1 nM), respectively.     

Beta-1,3-glucan inducible expression of prophenoloxidase-activating proteinase from eri-silkworm, Samia cynthia ricini

A cDNA clone encoding possible prophenoloxidase-activating serine protease (PAP) was isolated by screening the cDNA library from immunized larval fat body of the wild silkmoth, Samia cynthia ricini. The cDNA encodes a 438 amino acid open reading frame with a predicted 20 residue signal peptide. Samia PAP has high sequence similarity to Bombyx mori and Manduca sexta PAPs, which contain two amino terminal clip domains followed by a carboxyl-terminal catalytic domain. The expression of the gene was barely detectable in the fat body of naive larvae, but induced after injection of the larvae with beta-1,3-glucans or bacterial cells.     

The brain neurosecretory cells of the moth Samia cynthia ricini: Immunohistochemical localization and developmental changes of the Samia homologues of the Bombyx prothoracicotropic hormone and bombyxin

We produced mouse antisera against synthetic peptides corresponding to the sequences of the Samia cynthia ricini homologues of the Bombyx mori PTTH and bombyxin. Immunohistochemical analyses of the Samia cephalic neuroendocrine system using these antisera were performed to identify the neurosecretory cells (NSC) containing the PTTH and bombyxin homologues and to examine the developmental changes in their amounts in the NSC. The results show that the PTTH and bombyxin homologues are produced by two pairs of dorsolateral and 16 pairs of dorsomedial NSC of Samia brain, respectively, and both are transported to, and released from, the corpora allata. No clear-cut correlation was found between the fluctuation in the amount of immunoreactive substances in the brain NSC and the endocrinologically anticipated timings of PTTH secretion. From Samia brain extract, two forms of PTTH activity (∼30 kDa and ∼5 kDa) were resolved through Sephadex gel filtration. The ∼30 kDa and ∼5 kDa PTTH seem to represent the PTTH and bombyxin homologues, respectively. We discuss that the ∼30 kDa PTTH homologue is the true PTTH of Samia .     

Bombyxin: An Insect Brain Peptide that Belongs to the Insulin Family

Bombyxin is a 5 kDa secretory brain peptide that belongs to the insulin family. Bombyxin of the silkmoth Bombyx mori can induce adult development when injected into brain-removed dormant pupae of the saturniid moth Samia cynthia ricini by activating the prothoracic glands to synthesize and release ecdysone. Bombyx bombyxin has been shown to lower the concentration of the major haemolymph sugar, trehalose, and to elevate the trehalase activity in the midgut and muscles in Bombyx, but the doses required to be effective are higher than the amounts in the feeding larvae. The exact physiological function of bombyxin in Bombyx itself is therefore still obscure, but its insulin-like structure suggests it has important roles. Bombyxin comprises a mixture of highly heterogeneous molecular forms whose amino acid sequences have 40% identity with human insulin. The Bombyx bombyxin gene encodes a precursor consisting of the signal peptide, B chain, C peptide, and A chain, in that order from the N terminus. So far, 32 bombyxin genes have been identified in Bombyx, and they are classified into 7 families, A to G, according to their sequence similarity. The bombyxin genes have no introns and cluster in unique distribution patterns. The gene arrangement in the cluster has been classified into three categories: gene pairs, gene triplets, and single genes. Nucleotide sequence analysis indicates that equal and unequal crossings-over and duplications may have generated these unique distribution patterns. The Bombyx bombyxin genes are expressed predominantly in the brain and at low levels in a number of other tissues. Genes of all 7 families are expressed in four pairs of the medial neurosecretory cells of the brain. Detailed examination indicated that only a limited number of genes in the A, B and C family members are expressed and that their expression shows a gene-arrangement-dependent pattern.     

Functional analysis of the SGNP I in the pupal diapause of the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae)

Helicoverpa assulta suboesophageal ganglion neuropeptide I (Has-SGNP I) is a 24-amino acids peptide amide, which shows 62.5% similarity with the diapause hormone of Bombyx mori (Bom-DH). It has been demonstrated that embryonic diapause is induced by DH in B. mori. Injection of synthetic amidated Has-SGNP I terminated pupal diapause in a dose-dependent manner. Therefore, Has-SGNP I might be referred to a "diapause termination hormone" in H. assulta (Has-DTH). The maximal dose of Has-DTH for diapause termination was 1.0 microg and the half-maximal dose 0.4 microg. The time required for diapause termination of Has-DTH was 2-3 days longer than that of 20-hydroxyecdysone. During the pupal stage, DTH mRNA content in the SGs of nondiapausing pupae was always higher than in diapausing pupae using the combined method of quantitative RT-PCR and Southern blot. DTH gene also expressed at a low level while diapausing pupae were chilled at 4 degrees C, but increased rapidly and largely after being transferred to 25 degrees C. Using a competitive ELISA, Has-DTH-like immunoreactivity in the haemolymph showed the same pattern as that of Has-DTH gene expression. Those results indicated that Has-DTH gene expression was related to diapause development and could be activated by low temperature. Has-DTH might be useful to elucidate the mechanism of diapause termination in pupal diapause species.     

家蚕蛹营养成分及其开发利用研究进展

总结了家蚕BombyxmoriL .蛹的蛋白质、脂肪酸和几丁质等营养成分含量及其特点。系统介绍了家蚕蛹蛋白、脂肪酸和几丁质等成分的提取技术和相关生产工艺。概述了家蚕蛹作为保健食品与饲料原料开发状况和食用安全性。并对家蚕蛹营养成分利用与产品开发前景进行了展望。     

He—Ne激光辐照野桑蚕生物学效应研究

采用一定量He-Ne激光辐照野桑蚕催青卵及蚕肾,并与家蚕杂交,对其子代的蛋白质和酯酶同工酶进行电泳分析,结果发现谱带数目及活性有变化。另外,茧质调查和丝质调查与对照相比也有差异,并发现丝长和纤度特好的变异个体,显示He-Ne激光辐照对野桑蚕生理生化性状有一定影响。     

Prothoracicotropic Hormone Bioassay: Pupal-Adult Bombyx Assay

Blockage of adult development by brain removal and its resumption by application of the prothoracicotropic hormone (PTTH) were studied using pupae of a racial hybrid J-122 × C-115 of Bombyx mori . A log-linear dose-response relationship was obrained after injection of a PTTH solution. The Bombyx -unit of PTTH has been defined from this dose-response curve.     

大肠杆菌及聚肌胞核苷酸对柞蚕、家蚕蛹诱导产生溶菌酶、抗菌肽及凝集素的动力学

大肠杆菌D₃₁诱导柞蚕蛹产生抗菌多肽。同时,溶菌酶和凝集素活性都比诱导前有明显增高.其活力高峰、抗菌多肽在第7天左右、溶菌酶在第5天,而凝集素在第3天即达最高水平。不同品种的柞蚕蛹,经诱导产生的三种活性物质其活力差异不明显,但741、河四和小混品种中的抗菌多肽P_9A及P_9B组成比例较高。上述三种活性物质的诱导变化与性别有关,雄性高于雌性。比较了柞蚕蛹和家蚕经细菌诱导后上述三种活性物质的变化,家蚕凝集素活力很低,诱导后活力增高不明显。抗菌活力及溶菌酶活力的提高程度柞蚕也高于家蚕。聚肌胞核苷酸(Poly I:C)也能诱导两种蚕产生抗菌多肽及溶菌酶,但活力提高的显著程度都不及大肠杆菌诱导,凝集素活力变化也不显著。     

家蚕(Bombyx mori)主要血浆蛋白质及其基因表达的研究

本文用SDS-PAGE法观察不同发育阶段蚕血液中主要血浆蛋白质sp、30KP浓度的变化;从不同发育阶段的蚕脂肪体提取RNA和poly(A)~+-RNA,在兔网织红细胞系作体外翻译并检测翻译产物。结果表明,5龄蚕脂肪体mRNA合成蛋白质的速率为初蛹的2倍;5龄及初蛹脂肪体30KP mRNA活性的发育变化与其相应蛋白质在血液中的浓度变化一致;sp-1在5龄幼虫脂肪体内的表达及卵黄原蛋白(Vg)在蚕蛹脂肪体内的表达具有雌特异性,其表达和性特异性大体是在前翻译水平被调节的。     

Comparative genetic diversity and genetic structure of three Chinese silkworm species Bombyx mori L. (Lepidoptera: Bombycidae), Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan (Lepidoptera: Saturniidae)

The genetic diversity and genetic structure of three Chinese silkworm species Bombyx mori L., Antheraea pernyi Guérin-Meneville and Samia cynthia ricini Donovan were comparatively assessed based on RAPD markers. At the species level, A. pernyi and B. mori showed high levels of genetic diversity, whereas S. cynthia ricini showed low level of genetic diversity. However, at the strain level, A. pernyi had relatively highest genetic diversity and B. mori had lowest genetic diversity. Analysis of molecular variance (AMOVA) suggested that 60% and 72% of genetic variation resided within strains in A. pernyi and S. cynthia ricini, respectively, whereas only 16% of genetic variation occurred within strains in B. mori. In UPGMA dendrogram, individuals of A. pernyi and B. mori formed the strain-specific genetic clades, whereas those of S. cynthia ricini were distributed in a mixed way. The implications of these results for the conservation and utilization in breeding programs of three silkworm species are discussed.     

Expression, purification and characterization of human GM-CSF using silkworm pupae (Bombyx mori) as a bioreactor

To date, many recombinant proteins have been expressed in Bombyx mori cells or silkworm larvae, apart from in pupae. Silkworm pupae may be more suitable for the expression of heterologous proteins as a bioreactor. If maintained at an appropriate temperature, silkworm pupae could be inoculated with recombinant baculovirus for the expression of a protein of interest. In this study, human granulocyte-macrophage colony-stimulating factor was successfully expressed in silkworm pupae using B. mori nucleopolyhedrovirus, purified and characterized with respect to its physico-chemical properties. The target protein expressed had an apparent molecular mass of 29 kDa and an isoelectric point of 5.1. The protein was purified using three chromatographic steps with a final recovery of 10.3%. Finally, approximately 3.5mg of the protein was obtained with a biological activity of up to 8.4 x 10(6) cfu mg(-1). The results of this study suggest that silkworm pupae represent a convenient and low-cost bioreactor for the expression of heterologous proteins.     

注射大肠杆菌或超声波诱导家蚕及蓖麻蚕产生抗菌物质的比较研究

本文报道:1.蓖麻蚕hilosamia cynthia ricini、家蚕Bombyx mori及柞蚕Antheraea pernyi注射大肠杆菌Escherichia coli或超声波处理均能诱导血淋巴产生抗菌物质。同一蚕种诱导产物相同,不同蚕种间差异明显。2.家蚕不同发育阶段的个体诱导产生的抗菌物质基本相同;不同性别的家蚕蛹的诱导产物的相对量有差异;不同品种家蚕蛹对诱导的应答潜伏期也不尽相同。3.注射聚肌胞核苷酸(PolyI:C)诱导家蚕血淋巴产生一种明显地不同于其它诱导源诱导的抗菌物质,此物质在酸性聚丙烯酰胺凝胶电泳中泳动速度较快。 本研究的所有结果均表明,诱导绢丝昆虫产生抗菌物质的诱导源是非专一性的,即诱导源与诱导产物之间无对应的关系。     

Study of protein conformation and orientation in silkworm and spider silk fibers using Raman microspectroscopy

Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the beta-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters P2 and P4 of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of P2 and P4 using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the C=O stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the beta-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of P2 and P4 obtained are equal to -0.36 +/- 0.03 and 0.19 +/- 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of P2 and P4 of -0.32 +/- 0.02 and 0.13 +/- 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the beta-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of P2 and P4 using the information entropy theory. For the three types of silk, the beta-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the beta-sheets are nearly Gaussian functions with a width of 32 degrees and 40 degrees for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments.     

家蚕抗菌肽CMIV基因结构改造及表达产物的研究

参照天然抗菌肽ＣＭＩＶ组分的氨基酸序列,作了近５０％的改动,根据大肠杆菌偏爱的密码子,设计并人工合成了抗菌肽基因片段．将人工合成的抗菌肽类ＣＭＩＶ基因先重组到测序载体ｐＵＣ１１８上,经过序列分析,发现克隆于载体ｐＵＣ１１８上的基因片段与设计的序列完全一致．再将该基因片段重组到表达载体ｐＥＴ２８（ａ）上,抗菌肽以融合蛋白的形式表达．融合蛋白经镍－金属离子胶亲和层析纯化后,再用ＣＮＢｒ裂解,最终产物具有与天然抗菌肽相同的生物学活性     

The cDNA-structure of the prothoracicotropic hormone (PTTH) of the silkmoth Hyalophora cecropia.

The structure of the prothoracicotropic neurohormone (PTTH) of the silkmoth Hyalophora cecropia was elucidated at the cDNA level. The identified cDNA of 803 nt, which is over 90% identical with the corresponding part of the Samia cynthia ricini PTTH gene, encodes a preprohormone of 240 amino acids. Presence of proteolytic cleavage sites indicates that the preprohormone is split into a signal peptide, an intercalated peptide (64 residues), and the PTTH monomer (125 residues). Preprohormones of H. cecropia, S. c. ricini, Antheraea pernyi, and Bombyx mori diversified considerably in all these parts, indicating that the evolution of PTTH is unusually fast. Since a similarly rapid, and concerted evolution of the corresponding receptor is unlikely, the PTTH activity probably depends on the conservation of relatively few amino acids allowing proper molecular folding.