Molecular cloning of pheromone biosynthesis activating neuropeptide in silkworm, Bombyx mori

Pheromone biosynthesis activating neuropeptide (PBAN) is a suboesophageal ganglion secretory polypeptide of insect, which activates the pheromone gland to produce sex pheromone biosynthesis in female silkworm, Bombyx mori. A Bombyx genomic library was screened by the method of plaque hybridization using the 32P-labeled BomDH cDNA as a probe. The genomic sequence encoding PBAN has been cloned and its structure is analyzed. The PBAN gene comprises two exons interspersed by a single intron 697 bp in length. Preceding the PBAN amino acid sequence is a 32-amino acid sequence containing two FXPRL amide peptides, which are α-SGNP (Ile-Ile-Phe-Thr-Pro-Lys-Leu) and β-SGNP (Ser-Val-Ala-Asn-Pro-Arg-Thr-His-Glu-Ser-Leu-Glu-Phe-Ile-Pro-Arg-Leu), which is followed by a Gly-Arg processing site. Immediately, after the PBAN amino acid sequence is a Gly-Arg processing site and a FXPRL amide peptide γ-SGNP (Thr-Met-Ser-Phe-Ser-Pro-Arg-Leu). It is suggested that besides PBAN, 7-, 8-, and 17-residue amidated peptides wer     

Interneurons sensitive to female pheromone in the deutocerebrum of the male silkworm moth, Bombyx mori

ABSTRACT. In response to minute quantities of female sex pheromone, the male silkworm moth, Bombyx mori L., walks upwind to locate the odour source. The axons of antennal receptors specific for the two known components of the pheromone terminate in the deutocerebrum. In this study, single interneurons were recorded extracellularly in the deutocerebrum of the male silkworm moth. Responses were characterized as the antennae were presented with puffs of clean air, or air containing either or both components of the female pheromone, bombykol and bombykal. An apparatus is described which added bombykol or bombykal to a constant air stream flowing over the antenna. Most units (87%) showed qualitatively different responses to bombykol and bombykal. A majority of the pheromone-sensitive units (65%) also showed mechanosensory responses to air puffs. Two units were recorded which were slightly inhibited by either bombykol or bombykal alone, but were excited by a mixture of the two.     

家蚕Caspase家族基因 BmCaspase-X 的克隆与功能分析

【目的】本研究旨在克隆获得家蚕 Bombyx mori Caspase家族基因,并通过RNAi技术初步分析其在家蚕细胞水平细胞凋亡中的功能。【方法】 用cDNA末端快速扩增方法（rapid amplification of cDNA ends, RACE）克隆家蚕Caspase家族一个新基因,用RNAi和流式细胞术初步分析该基因在家蚕细胞凋亡中的功能。【结果】将克隆获得的家蚕Caspase家族基因命名为 BmCaspase-X,其全长为2 105 bp,开放阅读框长为1 494 bp,编码497 aa,分子量为57.8 kDa,等电点为5.29。BmCaspase-X含有Caspase特有的结构位点。系统进化分析表明,BmCaspase-X与家蚕ICE同其他昆虫Caspase-4同源物先聚在一起,再与具有长前体结构域的昆虫其他因子聚为一类。RNAi初步结果显示该基因干扰后没有出现明显的细胞凋亡。【结论】BmCaspase-X具有典型Caspase特征,属于Caspase家族成员。目前RNAi实验结果表明 BmCaspase-X 未能引起家蚕细胞凋亡的改变。本文为进一步研究该基因的功能奠定基础。     

Functional analysis of Dicer-2 gene in Bombyx mori resistance to BmNPV virus

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen in silkworm, and the molecular mechanism of B. mori defense to BmNPV infection is still unclear. RNA interference (RNAi) is well-known as an intracellular conserved mechanism that is critical in gene regulation and cell defense. The antiviral RNAi pathway processes viral double-stranded RNA (dsRNA) into viral small interfering RNAs that guide the recognition and cleavage of complementary viral target RNAs. In this study, a Dicer-2 (Dcr2) gene was identified in B. mori and its antiviral function was explored. Dcr2 messenger RNA (mRNA) expression was the highest in hemocytes and expressed in all stages of silkworm growth. After infection with BmNPV, the expression of Dcr2 mRNA was significantly increased after infection in midgut and hemocytes. The expression of Dcr2 was significantly upregulated by injecting dsRNA (dsBmSPH-1) into silkworm after 48 hr. Knocking down the expression level of Dcr2 using specific dsRNA in silkworm, which modestly enhanced the production of viral genomic DNA. Our results suggested that the Dcr2 gene in B. mori plays an important role in against BmNPV invasion.     

家蚕信息素结合蛋白BmPBP2和BmPBP3基因的初步鉴定及表达分析

嗅觉对昆虫的生存、繁殖等起着重要的作用。依据家蚕Bombyx mori全基因组序列设计特异引物,扩增得到了两个信息素结合蛋白BmPBP2和BmPBP3基因的cDNA片段。结合已报道的家蚕信息素结合蛋白BmPBP1和两个普通气味结合蛋白BmGOBP的信息,对其基因结构分析表明,这5个基因均由3个外显子组成,具有保守的外显子/内含子边界和典型的6个Cys残基,且3个PBP基因在基因组上串联分布。序列同源性分析表明,BmPBP2和BmPBP3与烟草天蛾的PBP2和PBP3的同源性高达69%和63%。半定量RT-PCR分析结果显示,BmPBP2和BmPBP3基因在成虫触角中特异表达,且雌雄表达水平相当。这些结果表明BmPBP2和BmPBP3可能起着性信息素识别的作用。     

Molecular mechanisms underlying sex pheromone production in the silkmoth, Bombyx mori: characterization of the molecular components involved in bombykol biosynthesis

Many species of female moths produce sex pheromones to attract conspecific males. To date, sex pheromones from more than 570 moth species have been chemically identified. Most moth species utilize Type I pheromones that consist of straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester and usually with several double bonds. In contrast, some moth species use unsaturated hydrocarbons or hydrocarbon epoxides, classified as Type II lepidopteran pheromones, as sex pheromones. Studies over the past three decades have demonstrated that female moths usually produce sex pheromones as multi-component blends where the ratio of the individual components is precisely controlled, thus making it possible to generate species-specific pheromone blends. As for the biosynthesis of Type I pheromones, it is well established that they are de novo synthesized in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways. However, as many of the molecular components within the PG cells (i.e., enzymes, proteins, and small regulatory molecules) have not been functionally characterized, the molecular mechanisms underlying sex pheromone production in PG cells remain poorly understood. To address this, we have recently characterized some of the molecules involved in the biosynthesis of the sex pheromone bombykol in the silkmoth, Bombyx mori. Characterization of these, and other, key molecules will facilitate our understanding of the precise mechanisms underlying lepidopteran sex pheromone production.     

家蚕丙氨酸转氨酶的纯化与鉴定

应用细胞匀浆,硫酸铵分段盐析,DEAE-纤维素柱层析和羟基磷灰石柱层析等方法,从家蚕后部丝腺中成功地分离制备了高纯度的丙氨酸转氨酶,经聚丙烯酰胺凝胶电泳分析鉴定,本法制备的丙氨酸转氨酶已达到均一的纯度。     

家蚕性别决定研究进展

作为模式生物和经济昆虫,家蚕的性别决定向来受到广泛的关注,近年来更得到深入的研究.主要在分子水平上概述了国内外有关家蚕性染色体方面最新的研究状况,通过对比,探讨了家蚕的几个可能决定性别的基因及其作用过程,以及可能的性别决定靶基因,并就目前性别决定研究现状中的几个突出的问题进行了探讨.     

The Bombyx mori sex pheromone biosynthetic pathway is not mediated by cAMP

In most moths, sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN). How the extracellular PBAN signal is turned into a biological response has been the focus of numerous studies. In the classical scheme of signal transduction, activated G proteins relay the extracellular signal to downstream effector molecules such as calcium channels and adenylyl cyclase. The role of calcium in PBAN signaling has been clearly demonstrated, but the possible involvement of cAMP is not as straightforward. While cAMP has been shown to be necessary for PBAN signaling in most heliothine species, there has been no definitive demonstration of its role in Bombyx mori. To address this question, we used degenerate RT-PCR to clone two Gs subunits, designated P50Gs1 and P50Gs2, from B. mori pheromone gland (PG) cDNAs. The two Gs proteins were expressed in all tissues examined and were not up-regulated in accordance with adult eclosion. Even though two bands corresponding to the approximate molecular weights of P50Gs1 and P50Gs2 were detected in PG homogenates, the Gs antagonist, NF449, had no effect on sex pheromone production. Furthermore, no changes in the intracellular cAMP levels were detected following PBAN stimulation.     

Termination of sex pheromone production in mated females of the silkworm moth

A mating duration of more than 6 h was necessary to permanently terminate the production of the sex pheromone (bombykol) in the silkworm moth, Bombyx mori L. (Lepidoptera: Bombycidae), although the female formed a bursa copulatrix including a spermatophore and laid fertilized eggs even after mating for only 0.5 h. The 6-h mated female again produced bombykol if given an injection of synthetic pheromonotropic neuropeptide (PBAN), which is known to activate pheromone biosynthesis in a virgin female. Extracts of brain-suboesophageal ganglion (SG) complexes, which were removed from 6- and 24-h mated females, showed strong pheromonotropic activities. These results indicated that the pheromone gland of the mated female maintained its ability to biosynthesize bombykol; however, it could not produce pheromone due to a suppression of PBAN secretion from the SG. Furthermore, bombykol titers did not decrease after mating in females with a transected ventral nerve cord, even after the injection of a spermatophore extract, suggesting that the suppression of PBAN secretion was mediated by a neural signal and not by a substance in the spermatophore. The mated females accumulated (10E, 12Z)-10,12-hexadecadienoic acid, a precursor of bombykol biosynthesis, in their pheromone glands as did decapitated females. © 1996 Wiley-Liss, Inc.     

家蚕抗核型多角体病分子标记筛选

对家蚕抗核型多角病毒病以不同的杂交方式,构建3种近等基因系,用RAPD技术筛选出抗病主基因连锁分子标记OPA-18700和感病连锁分子标记OPY-11400。同时在F2群体中验证了抗性分子标记的有效性。     

Effect of the pheromone biosynthesis activating neuropeptide on sex pheromone biosynthesis in Spodoptera littoralis isolated glands

The control of Spodoptera littoralis sex pheromone biosynthesis has been investigated with synthetic pheromone biosynthesis activating neuropeptide (PBAN) and different labeled tracers using an in vitro isolated gland system. Responsiveness of the glands to PBAN stimulation was impaired by careless tissue manipulation. The fact that PBAN is active in the isolated gland system suggests that this might be a target organ for this peptide in S. littoralis. As reported previously with Br-SOG extracts and intact females, label incorporation into the pheromone increased in glands treated with PBAN from all the precursors tested. However, the formation of labeled intermediates from d₅E11–14:Acid also occurred in glands incubated in the absence of the peptide, but the amounts of d₅Z9, E11–14:Acid were lower in PBAN treated glands than in controls. These results indicate that PBAN controls pheromone biosynthesis in S. littoralis by regulating the reduction of acyl moieties. © 1994 Wiley-Liss, Inc.     

Identification of a PP2A gene in Bombyx mori with antiviral function against B. mori nucleopolyhedrovirus

Ser/Thr protein phosphatase 2A (PP2A) is one of the type 2 protein phosphatases, which is required for many intracellular physiological processes and pathogen infection. However, the function of PP2A is unclear in silkworm, Bombyx mori. Here, we cloned and identified BmPP2A, a PP2A gene from B. mori, which has two HEAT domains and a high similarity to PP2A from other organisms. Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut, and that Bombyx mori nucleopolyhedrovirus (BmNPV) infection induces down‐regulation of BmPP2A expression. Furthermore, up‐regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication. In contrast, down‐regulation of BmPP2A via RNA interference and okadaic acid (a PP2A inhibitor) treatment allowed robust BmNPV replication. This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A, a potential anti‐BmNPV mechanism, and a possible target for the breeding of silkworm‐resistant strains.     

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.     

家蚕微孢子虫感染对家蚕海龟蛋白(Bmtutl)基因表达水平的影响

【目的】本研究旨在阐明家蚕微孢子虫Nosema bombycis感染不同时间对家蚕Bombyx mori幼虫不同组织中家蚕海龟蛋白(Bombyx Turtle, Bmtutl)基因表达水平的影响,为揭示家蚕微孢子虫的侵染机制奠定基础。【方法】利用生物信息学方法对家蚕海龟蛋白3种亚型Bmtutl-464, Bmtutl-519和Bmtutl-810的序列结构特征进行了分析;利用qPCR检测家蚕微孢子虫感染后12, 24, 48, 72, 96和120 h,家蚕幼虫中肠、血淋巴与脂肪体组织中Bmtutl-464, Bmtutl-519和Bmtutl-810基因表达水平的变化情况。【结果】家蚕海龟蛋白3种亚型的二级结构均主要由无规则卷曲、α螺旋、β转角和延伸链组成,其中无规则卷曲所占比例最高。但是PredictProtein分析发现,Bmtutl-464, Bmtutl-519和Bmtutl-810之间的蛋白/多核苷酸结合位点存在较大差异。qPCR结果表明,感染家蚕微孢子虫后,家蚕幼虫中肠、血淋巴与脂肪体组织中Bmtutl-464, Bmtutl-519和Bmtutl-810基因的整体表达处于被抑制状态,尤其在脂肪体中最为明显：Bmtutl-519和Bmtutl-810基因的表达在家蚕微孢子虫感染家蚕后的72 h开始受到显著抑制,特别是Bmtutl-519基因,其相对表达水平均不到对照的5.0%。【结论】家蚕海龟蛋白这3种亚型的序列结构特征存在较大差异,家蚕微孢子虫感染在一定程度抑制了家蚕幼虫中肠、血淋巴与脂肪体组织中Bmtutl-464, Bmtutl-519和Bmtutl-810基因尤其是Bmtutl-519的表达。结果说明,与其他两种家蚕海龟蛋白亚型相比,Bmtutl-519蛋白可能在家蚕微孢子虫侵染宿主的过程中起主要作用。