phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Transgenic silkworms can be useful for investigating the functions of genes in the post-genomic era. However, the common method of using a transposon as an insertion tool may result in the random integration of a foreign gene into the genome and suffer from a strong position effect. To overcome these problems, it is necessary to develop a site-specific integration system. It is known that phiC31 integrase has the capacity to mediate recombination between the target sequences attP and attB. To test the availability of site-specific integration in the silkworm, we first examined the efficiency of recombination between the target sites of the two plasmids in silkworm embryos and found that the frequency of recombination was very high. Then we constructed a host strain that possessed the target sequence attP using the common method. We injected the donor plasmid together with the phiC31 integrase mRNA into the embryos of the host strain and obtained positive lines. Structural analysis of the lines showed that site-specific integration occurred by recombination between the genomic attP site and the attB site of the donor plasmid. We can conclude from the results that phiC31 integrase has the ability to mediate the site-specific integration of transgenes into the silkworm chromosome.     

家蚕Bcl-2家族基因 BmBuffy 的克隆及表达分析

【目的】本研究旨在获得家蚕 Bombyx mori Bcl-2家族同源基因,并分析其在不同组织和发育阶段的时空表达模式及功能。【方法】用cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆家蚕Bcl-2家族基因BmBuffy,利用SSR分子标记连锁分析确定其染色体定位。同时,用RT-PCR和qPCR技术分析该基因在家蚕幼虫及变态期间不同组织中的表达。【结果】克隆获得全长家蚕Bcl-2家族同源基因 BmBuffy,证明该基因位于第 4号染色体上,开放阅读框长879 bp,编码292 aa,预测其分子量大小为32.4 kDa,等电点为9.94,且第130-231位氨基酸之间存在1个Bcl-2_like Superfamily结构域。系统进化发育树表明,其与黑腹果蝇Drosophila melanogaster的DmBuffy关系最近,氨基酸序列一致性为27%。BmBuffy在幼虫组织中的表达结果显示,其在马氏管中表达量最高,而且在不同组织变态期的关键时间点均有明显变化。【结论】家蚕BmBuffy具有Bcl-2家族典型结构域,BmBuffy定位于第 4号染色体上,BmBuffy在家蚕变态期的组织生理变化中起到一定作用。本文为进一步研究家蚕Bcl-2家族基因的功能奠定基础。     

家蚕鞣化激素基因对翅展及繁殖力的调控作用

[目的]昆虫鞣化激素(Bursicon)是由神经系统分泌的一种异源二聚体神经肽,对昆虫表皮鞣化、翅展等功能具有调控作用.本研究旨在探究鞣化激素基因与家蚕Bombyx mori翅发育及对繁殖力相关基因的关系,明确其对翅展和繁殖力的调控作用.[方法]采用RNAi技术,分别注射Bursicon基因的dsRNA(dsBmBur...     

Arginine kinase is highly expressed in a resistant strain of silkworm (Bombyx mori, Lepidoptera): Implication of its role in resistance to Bombyx mori nucleopolyhedrovirus

A gene encoding Bombyx mori arginine kinase (BmAK) has been indentified differentially expressed in the midguts of Bombyx mori strain NB which is resistant to nucleopolyhedrovirus (BmNPV), strain 306 which is susceptible to NPV and a near isogenic line BC(8) with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). In this study, we characterized the expression profiles of BmAK using RT-PCR and real-time quantitative PCR. The expression level of BmAK fluctuated in various developing stage and various tissue. Remarkably, the expression level of BmAK increased more than 10-fold 24 hours post inoculation (h p.i.) of NPV in strain NB and BC(8), while such increment was abraded in strain 306 although the basal expression level of BmAK in strain 306 was higher than that of strain NB and BC(8). Western blotting analysis using polyclonal antibody against BmAK verified such observation, and immunofluoresence analysis indicated for the first time that BmAK was mainly located to the cytoplasm or some structures in cytoplasm. These findings suggest that arginine kinase is involved in the antiviral process of Bombyx mori larvae against NPV infection.     

BAC selection of the large white butterfly,Pieris brassicae (Lepidoptera: Pieridae) containing orthologs of the silkworm,Bombyx mori (Lepidoptera: Bombycidae)

Applied Entomology and Zoology - We constructed a bacterial artificial chromosome (BAC) library from the large white butterfly, Pieris brassicae (L.) (Lepidoptera: Pieridae), which...     

Morphology of the Pylorus of Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae)

The pylorus is a segment of the hindgut of great morphological diversity, which is subdivided into the posterior interstitial ring, pyloric cone, and pyloric valve. There is no morphological information on Bombyx mori (L.) hybrids, which is an insect of economic interest to Brazil. Consequently, this study aimed to analyze the morphology of the pylorus of hybrid larvae of fifth instars of B. mori from crossbreeding of Chinese and Japanese races. These were anesthetized, and the pylorus was fixed and processed by light microscopy and scanning electron microscopy. The pylorus presented as a separate compartment, consisting of a simple epithelium, with large variation in cell morphology, covered on the luminal side by an intima with specializations in some areas, the spicules. The musculature was formed by bundles of striated muscle fibers in longitudinal and circular arrangement. These fibers were multinucleated with central and peripheral nuclei, operating in peristalsis. The morphology of the pylorus is a reflection of functional dynamism, and this study provided a better understanding of its physiology, which promotes the regulated movement of the bolus, preventing reflux and directing it towards the ileum in the formation of fecal pellets.     

Effects of Diflubenzuron on the Development of Bombyx mori (Lepidoptera: Bombycidae)

ABSTRACT This study was conducted to determine the effects of diflubenzuron (DFB) on the growth and development of the silkworm, Bombyx mori (L.). The DFB treatment extended abnormally the larval duration and affected negatively on larval spinning of the 5th instar. All the larvae treated with high DFB concentration (2.5 × 10^-1 and 2.5x 10^-1μg/μl) lost their spinning capability and finally died, whereas 62% of larvae treated with low DFB concentration (2.5 × 10^-1μg/μl)) spinned cocoon. The larval weights depended sensitively on the DFB treatment period rather than on the DFB concentration. The DFB treatment decreased the larval maturity less than 6% without regard to the concentration and treatment period. All the larva, when treated with DFB before the 5th days of the 5th instar, were not matured.     

Male meiosis in polyploid silkworms,Bombyx mori L. (Lepidoptera : Bombycidae)

Tetraploid and hexaploid silkworms, Bombyx mori L. (Lepidoptera Bombycidae) were induced by applying a cold shock to diploid and triploid eggs at the first cleavage stage. Male meiosis in the primary spermatocytes of these silkworms having a different ploidy was observed. In the polyploid cells, chromosome bridges, 2 for the tetraploid and 3 for the hexaploid, occurred between the newly formed daughter nuclei at telophase. Observation in the living spermatocytes showed that tetraploid cells needed a longer time than the diploid ones to complete the first meiotic division. The delay in the cell division may be responsible for the high sterility of the tetraploid males.     

Rab14 from Bombyx mori (Lepidoptera: Bombycidae) shows ATPase activity

Rab GTPases are essential for vesicular transport, whereas adenosine triphosphate (ATP) is the most important and versatile of the activated carriers in the cell. But there are little reports to clarify the connection between ATP and Rab GTPases. A cDNA clone (Rab14) from Bombyx mori was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein bound to [³H]-GDP and [³⁵S]-GTPγS. Binding of [³⁵S]-GTPγS was inhibited by guanosine diphosphate (GDP), guanosine triphosphate (GTP) and ATP. Rab14 showed GTP- and ATP-hydrolysis activity. The Km value of Rab14 for ATP was lower than that for GTP. Human Rab14 also showed an ATPase activity. Furthermore, bound [³H]-GDP was exchanged efficiently with GTP and ATP. These results suggest that Rab14 is an ATPase as well as GTPase and gives Rab14 an exciting integrative function between cell metabolic status and membrane trafficking.     

C-型咽侧体抑制素(AST-C)和促前胸腺激素释放激素(PTTH)基因在家蚕脑组织中的时空表达分析

【目的】咽侧体抑制素在昆虫体内具有重要的调控功能。本研究比较了家蚕Bombyx mori C-型咽侧体抑制素(allatostatin C,AST-C)与促前胸腺激素释放激素(prothoracicotropic hormone,PTTH)基因在不同发育阶段家蚕脑组织中转录表达的模式及其在脑组织的表达定位,以期为家蚕AST-C的功能研究提供重要的线索。【方法】利用脑组织芯片数据分析比较AST-C和PTTH基因在家蚕脑组织中的发育表达特征,RT-PCR验证其芯片数据,分析AST-C在不同发育阶段家蚕中枢神经系统中的表达模式,并用全组织包埋原位杂交技术对AST-C和PTTH在脑组织的表达进行定位。【结果】AST-C和PTTH在不同发育阶段家蚕脑组织中有相似的转录表达模式,且都在脑组织外侧一对神经分泌细胞中表达。【结论】AST-C可能与PTTH以相同的转录表达模式共同参与家蚕的变态发育调控。     

细菌感染后家蚕幼虫中肠差异表达基因的鉴定（英文）

作为遭遇各种微生物的前线, 昆虫的消化道在免疫反应中起到重要的作用。为了研究家蚕Bombyx mori肠道免疫反应, 我们利用基于ACP (annealing control primer)的反转录PCR技术, 从中肠组织中鉴定得到18个在经口器感染绿脓杆菌Pseudomonas aeruginosa 和金黄色葡萄球菌Staphylococcus aureus后的差异表达基因,并利用荧光定量PCR分析了其中4个基因在感染后24 h内的动态变化。结果表明： 肽聚糖识别蛋白-L1（PGRP-L1)和一个丝氨酸蛋白酶前体基因特异性地受绿脓杆菌感染后上调; 30kP蛋白酶A基因受绿脓杆菌和金黄色葡萄球菌2种细菌感染后上调。我们的研究鉴定了经口器感染后家蚕中肠中参与入侵细菌识别和免疫信号途径的基因, 可为对这些基因进一步的功能研究提供线索。     

Influence of Wolbachia infection on antimicrobial peptide gene expressions in a cell line of the silkworm,Bombyx mori (Lepidoptera: Bombycidae)

Wolbachia, a symbiotic bacterium found in a broad range of insects, manipulates host reproduction. In addition to reproductive alterations, Wolbachia may also modify the immune system of host insects to protect them from additional pathogenic infection. We hypothesized that Wolbachia exerts protective effects by activating antimicrobial peptide (AMP) genes. To test this hypothesis, we established immunocompetent cell lines derived from the silkworm, Bombyx mori, which were transinfected with two Wolbachia strains, wKue and wCauB, originating from lepidopteran insects and quantified the expression of four AMP genes, cecropin B, defensin B, attacin, and lebocin 3. The expression was measured in wKue-infected, wCauB-infected, and uninfected cells, before and after treatment with peptidoglycan (PGN) that mimicked a bacterial infection. A two-way ANOVA for each gene showed that both Wolbachia infection and PGN treatment significantly increased the gene expression and their interaction. When treated with PGN, wKue- and wCauB-infected cells showed higher expression of the four AMP genes than those in uninfected cells, suggesting that Wolbachia infection increased the ability of host cells to produce AMPs in response to immune stimulation with PGN. These observations suggest that the two Wolbachia strains have immune priming effects and may protect the host insects from a secondary infection.     

感染球孢白僵菌的家蚕免疫应答差异表达基因分析

【目的】筛选家蚕Bombyx mori应对白僵菌Beauveria bassiana侵染的应答基因, 以进一步研究家蚕抵御真菌侵染的分子机制。【方法】采用新一代Solexa高通量测序技术对感染白僵菌及未感染白僵菌的对照组家蚕进行了测序分析, 筛选差异表达基因; 结合生物信息学工具分析差异表达基因的功能注释、 分类及涉及的信号通路等; 应用荧光定量PCR技术验证10个基因的差异表达。【结果】通过测序和生物信息学分析共获得377个差异表达基因, 其中表达上调基因236个, 下调基因141个; KEGG通路分析表明, 各通路中既有表达上调的基因, 也有下调基因; 12个上调基因、 26个下调基因参与3个显著性富集的KEGG通路, 即核糖体、 氨酰tRNA生物合成和剪接体通路。定量PCR与测序结果显示, 溶菌酶、 热激蛋白、 谷胱甘肽S-转移酶、 肽聚糖识别蛋白等与免疫应激相关的蛋白基因均呈现表达上调。【结论】本研究筛选获得的差异表达基因, 特别是上调表达的基因可能与家蚕应对白僵菌侵染的应答机制有关, 其中与免疫应激相关的蛋白基因如溶菌酶、 热激蛋白、 谷胱甘肽S 转移酶、 肽聚糖识别蛋白基因等可能直接参与了家蚕对白僵菌的免疫识别和防御, 研究结果为从分子水平阐明家蚕抵御真菌侵染的防御机制和白僵菌对家蚕的致病机理提供新的依据。     

Pachytene mapping in the female silkworm,Bombyx mori L. (Lepidoptera)

Pachytene preparations of the chromosome complement of female larvae of Bombyx mori were improved to give a distinct chromomere pattern of the bivalents suitable for chromosome mapping. Six of the 28 bivalents are described and can be identified regularly in the bivalent complements, among them the bivalent containing the nucleolus organizer. The relative lengths of these bivalents compared with one another change during development of pachytene. In contrast to other members of the Lepidoptera there is no conspicuous heterochromatic W-chromosome, which corresponds to the female-specific heterochromatin body present in the nuclei of somatic tissues. This tissue-specific heteropycnosis indicates a different functional state of the responsible chromosome or chromosomal segment in germ line and somatic cells.     

Effect of fetal bovine serum on the enhancement of in-vitro cultivation of spermatocysts of the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae)

Like other Lepidoptera, the silkworm (Bombyx mori) has both nucleated eupyrene and anucleated apyrene sperm that are derived from the same spermatocysts. The former type is responsible for egg fertilization, while the function of the latter is still uncertain. Many hypotheses have been presented concerning the role of the apyrene sperm in mating and fertilization, but none is supported by a convincing experimental approach. The aim of the present study was to enhance the production of apyrene sperm in vitro by using different concentrations of fetal bovine serum (FBS), namely 20%, 30% and 40%, in the culture medium used for cultivating the naked spermatocysts isolated from the silkworm testes at 0 hr, 120 hr, and 192 to approximately 360 hr after the fourth molt. Cultivation of 0-hr spermatocysts was not successful. The development of spermatocysts into eupyrene and apyrene sperm bundles was slightly slower in vitro than in vivo. The overall growth percentage of both eupyrene and apyrene bundles was satisfactory when the spermatocysts were cultivated in TC-100 culture medium containing 30% FBS.     

Behavior of centrioles during meiosis in the male silkworm, Bombyx mori (Lepidoptera)

The behavior of centrioles during eupyrene and apyrene meiosis was examined in the silkworm, Bombyx mori , by transmission electron microscopy and indirect immunofluorescence for tubulin. In eupyrene spermatocytes the centrioles, accompanied by axonemes, attached temporarily to the nucleus at diplotene, then detached from the nucleus in diakinesis. After the separation, a beret-shaped structure consisting of a double membrane covered the proximal region of the pair of centrioles. The structure disappeared after breakdown of the nuclear membrane. The centriole, with the axoneme, reattached to the nucleus at telophase I. The process was repeated during meiosis II until the centrioles maintained their nuclear attachment in newly developed spermatids. In stark contrast to their eupyrene counterparts, apyrene spermatocytes were conspicuously devoid of any attachment of the centrioles to the nucleus. These eupyrene-specific and apyrene-specific relationships were consistently and repeatedly found between the nuclear membrane and centrioles, giving rise to suspicion that the behavioral phenomena may be related to differentiation of the dimorphic sperm types.     

Influence of mulberry cultivars Morus spp. on the production and quality of silkworm Bombyx mori L. (Lepidoptera: Bombycidae) cocoons

The success of sericiculture greatly depends on the availability of mulberry cultivars with agronomic characteristics that maximize the production and quality of silkworm, Bombyx mori L., cocoons. The effect of the cultivars Miura (standard) and Korin, and the hybrids SM 14, SM 63, SK 1, SK 4 and FM 86 was evaluated on silkworm development, and on cocoon production and quality, at the Instituto Agron?mico do Paraná, IAPAR, in Londrina, PR, Brazil (23 +/- 3 degrees C; 61 +/- 8% RH). The experiment was arranged in a randomized block design with four replicates. Larvae feeding either on 'SK 4' or on 'Korin' showed a higher weight compared to that of larvae feeding on 'Miura'. Cocoon weight (both sexes) and pupae weight (male) obtained with larvae fed on SK 4 were higher compared with that of insects that fed on Miura; there was no difference among the remaining treatments. Silk net weight was independent of the cultivar used. Cocoon shell weight was similar amongst insects raised on 'Miura' and on the other mulberry materials. The percentage of cocoon spinning was higher than 90% in all treatments, with the spinning concentrating in the first two days. The proportion of first grade cocoons varied from 80% (SK4) to 90%. The results obtained with the studied cultivars were either similar or superior to that of the standard cultivar Miura with emphasis to 'SK 4', thus showing the potential use of these cultivars in sericulture in the state of Paraná.     

家蚕醛氧化酶基因（BmAOXs）的鉴定与表达分析

醛氧化酶（aldehyde oxidases, AO, EC 1.2.3.1）是属于钼-黄素酶（molybdo-flavoenzymes, MFEs）家族的一类蛋白酶。为了探讨该酶在家蚕Bombyx mori中的功能, 本研究对家蚕醛氧化酶基因（BmAOXs）家族进行了鉴定和分析。以其他物种AO基因序列检索家蚕全基因组数据库, 获得了8个BmAOX候选基因, 均具有醛氧化酶保守的功能域。进化分析表明, BmAOX与其他昆虫AO聚为一簇。RT-PCR分析结果显示: BmAOX1, BmAOX2, BmAOX3, BmAOX5具有很强的组织特异性; 而BmAOX4, BmAOX6, BmAOX7, BmAOX8则在蛹和成虫的多个组织中均有表达, 提示它们在家蚕生理代谢活动中起重要作用。利用Native PAGE和活性染色方法, 对BmAOX编码的蛋白进行检测, 结果表明: 家蚕蛹中有5种有活性的醛氧化酶, 而成虫中有6种, 各组织中均有有活性的BmAOX, 只是种类和活性水平有所不同。本研究结果为深入探讨BmAOX家族的功能奠定了基础。     

Modulation of leucine absorption in the larval midgut of Bombyx mori (Lepidoptera, Bombycidae).

In the larval midgut of Bombyx mori a K(+)-dependent transporter for leucine and amino acids with a hydrophobic side chain is responsible for the absorption of most essential amino acids. We investigated if a modulation of its activity occurred as a result of starvation or after hormonal treatments. We measured amino acid uptake in brush border membrane vesicles (BBMV) purified from the anterior-middle (AM) and posterior (P) regions of the midgut in fifth instar larvae. Silkworms were either starved or topically treated with low dosages of fenoxycarb, a molecule often used as a juvenile hormone mimic. The maximal uptake value of K(+)-driven leucine transport was increased in BBMV of AM- and P-midgut regions of starved larvae. The initial uptake rates of serine and glutamine, two amino acids transported by the same cotransporter as leucine, were also increased. Leucine kinetics proved that V(max) was the kinetic parameter modified by starvation in both midgut regions. Topical applications of fenoxycarb at a dose of 2.5 fg/larva immediately after the fourth ecdysis, induced an increase of leucine initial uptake rates and of intravesicular accumulation of leucine in both AM- and P-BBMV. Kinetic analysis of leucine uptake indicated again that V(max) was increased in BBMV from both midgut regions in treated larvae.