The nucleotide sequence of a major glycine transfer RNA from the posterior silk gland of Bombyx mori L.

The nucleotide sequence of tRNA1Gly isolated from the posterior silk gland of Bombyx mori has been determined. This transfer RNA is present in high amounts in the posterior silk gland during the fifth larval instar. It has a GCC anticodon, capable of decoding a major glycine codon in the fibroin messenger RNA, GGU. Structural features of Bombyx tRNA1Gly and its homology to other eukaryotic glycine tRNAs are discussed.     

Temporal changes in DNA synthesis of prothoracic gland cells during larval development and their correlation with ecdysteroidogenic activity in the silkworm, Bombyx mori

DNA synthesis in prothoracic gland cells of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU), and its developmental changes during the 3rd, 4th, and last larval instars were examined. During the early stages of both the 3rd and 4th larval instars, a dramatic increase in the number of DNA-synthesizing cells of the prothoracic glands was detected. However, during the latter stages of each instar, the number of DNA-synthesizing cells greatly decreased. The determination of glandular protein content showed that dramatic increases occurred during the latter stages of each larval instar. Comparison of changes in prothoracic gland cell DNA synthesis with ecdysteroidogenic activity showed that the increase in DNA synthesis precedes ecdysteroidogenesis. The cellular mechanism underlying changes in prothoracic gland cell DNA synthesis during the last two larval instars was further analyzed by determining the in vitro DNA synthesis of the glands, their responsiveness to hemolymph growth factors, and changes in the growth-promoting activity of hemolymph during development. It was found that both growth factors and the responsiveness of the prothoracic gland cells to growth factors from hemolymph may play roles in regulating DNA synthesis of gland cells.     

Stage-dependent effects of 20-hydroxyecdysone on DNA synthesis of corpus allatum cells in the silkworm,Bombyx mori

DNA synthesis in cells of the corpus allata (CA) of the silkworm, Bombyx mori, was studied immunocytochemically after in vivo labeling with 5-bromo-2'-deoxyuridine (BrdU); developmental changes during the 3rd, 4th, and last larval instars and effects of 20-hydroxyecdysone treatment were examined. During both the 3rd and 4th larval instars, the number of DNA-synthesizing cells fluctuated, and relatively low levels were observed during the middle stages. On day 0 of the last larval instar, the number of DNA-synthesizing cells per gland was 9.2, which then increased on day 1 and remained at levels ranging from 12.9 and 16.9 cells per gland. A major peak level (28 BrdU-labeled cells per gland) occurred on day 8, two days after larvae entered the wandering stage. When last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 0 or 1, the number of DNA-synthesizing cells dramatically decreased to very low levels and these low levels were maintained throughout the remainder of the instar. However, no effect was observed when last instar larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves starting on day 3, indicating the stage-specific action of 20-hydroxyecdysone. The mechanism by which 20-hydroxyecdysone treatment inhibits DNA synthesis of CA cells was further examined by using continuous in vitro BrdU labeling for a 2-day incubation. It was found that the decrease in responsiveness of DNA synthesis of CA cells of 20-hydroxyecdysone-treated larvae to stimulation by growth factors from hemolymph may have been, at least in part, responsible for the indirect inhibitory effects of 20-hydroxyecdysone.     

Cloning of mRNA sequence coding for sex-specific storage protein of Bombyx mori

A major plasma protein, referred to as SP 1, exhibits sex- and stage-specific expression during the larval development of the silkworm, Bombyx mori. We have isolated a plasmid clone bearing a part of mRNA sequence coding for SP 1 and quantitated the amount of SP 1 mRNA by means of RNA blot hybridization using the cDNA probe. The developmental change in the amount of SP 1 mRNA in the fat body closely reflected that of the hemolymph concentration of SP 1, indicating that the biosynthesis of SP 1 is regulated in a sex- and developmental stage-specific fashion at the level of mRNA.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.     

更换饲料对家蚕幼虫肠道细菌组成的影响及关键物种与产茧性能的相关性

【目的】本研究探讨使用人工饲料部分替代桑叶饲育后家蚕Bombyx mori幼虫肠道细菌群落的变化情况,并分析人工饲料替代桑叶的不同饲育模式下肠道关键细菌与蛹重和茧层量的相关性,为人工饲料部分替代法饲育家蚕的实际应用提供理论依据。【方法】采用全龄桑叶育(Mul1-5)、1-2龄人工饲料育+3-5龄桑叶育(Art1-2)、1-3龄人工饲料育+4-5龄桑叶育(Art1-3)、1-4龄人工饲料育+5龄桑叶育(Art1-4)和全龄人工饲料育(Art1-5) 5种饲养模式饲养家蚕幼虫,统计5种饲养模式下家蚕的全茧量、茧层量及蛹重;收集不同饲养模式下家蚕5龄幼虫肠道样品,通过高通量测序技术分析其肠道菌群的组成和多样性差异,采用Spearman相关性热图分析肠道细菌与蛹重和茧层量的相关性。【结果】人工饲料与桑叶不同搭配的饲育模式对家蚕茧质有显著的影响,Mul1-5和Art1-5的全茧量最高,二者无显著性差异,Art1-2其次; Mul1-5茧层量最高,Art1-2其次,Art1-5茧层量最低。在不同饲育模式下,家蚕肠道细菌多样性和组成显著不同：在门水平上,Art1-2和Mul1-5的细菌组成最相近,优势菌主要是变形菌门(Proteobacteria)和厚壁菌门(Firmicutes),而Art1-3, Art1-4和Art1-5的细菌组成最相近,变形菌门丰度随着人工饲料饲养时间的增加逐渐增加,厚壁菌门的丰度则呈现相反的趋势;在属水平上,家蚕肠道优势菌属有假单胞菌属Pseudomonas、肠球菌属Enterococcus、鞘脂单胞菌科未分类菌属Sphingomonadaceae unclassified及罗尔斯通菌属Ralstonia等。不同饲育模式下家蚕肠道菌属丰度差异最大的是肠球菌属,Art1-2组中肠球菌属丰度最高(40.9%),随着人工饲料饲养时间的延长,丰度逐渐降低,在Art1-5中仅为0.02%。家蚕肠道菌群大部分关键物种均与茧质呈一定的相关性：柯克斯体属Coxiella与蛹重呈显著正相关,与茧层量呈显著负相关;葡萄球菌属Staphylococcus与蛹重呈显著负相关;肠球菌属与茧层量呈显著正相关。【结论】饲育模式Art1-2最接近于Mul1-5,可为人工饲料部分替代桑叶饲育法饲育家蚕提供参考。人工饲料部分替代桑叶后家蚕肠道菌群结构发生了显著变化,肠球菌属丰度的显著降低及假单胞菌属的显著增加可能与全龄人工饲料养家蚕体质弱有关;一些关键细菌与家蚕蛹重和茧层量存在显著相关性,其影响机制需要进一步研究。     

Fibroin silk proteins from the nonmulberry silkworm Philosamia ricini are biochemically and immunochemically distinct from those of the mulberry silkworm Bombyx mori

Silk proteins were isolated from the cocoons of the nonmulberry silkworm, Philosamia ricini. Three polypeptides of 97, 66, and 45 kDa were identified. The 66-kDa molecule represented sericin, whereas the 97-kDa and the 45-kDa polypeptides linked together through a disulfide bond constituted the fibroin protein. Antibodies raised against the 97-kDa P. ricini fibroin heavy chain reacted specifically with this molecule and did not recognize fibroin heavy chain from another nonmulberry silkworm, Antheraea assama or from the mulberry silkworm, Bombyx mori, suggesting the presence of P. ricini species-specific determinants in this heavy chain. Antibodies generated against fibroin light chain of P. ricini also showed similar reactivity pattern. Immunoblot analysis with proteins isolated from the silk glands of P. ricini at different stages of larval development showed that the expression of fibroin heavy chain was developmentally and spatially regulated. The protein was most abundant in the 5th instar larva, and could be detected in the middle and the posterior but not the anterior silk glands. The amino acid composition of the 97-kDa fibroin protein showed abundance of glutamic acid and did not contain (Gly-Ala)(n) motifs, a characteristic feature of B. mori fibroin heavy chain. Our study reveals significant differences between the nonmulberry silkworm P. ricini and the mulberry silkworm B. mori in the biochemical composition and immunochemical characteristics of fibroin heavy chain. These differences might be responsible for the differences seen in the quality of silk produced by these two silkworms.     

Tissue-specific regulation of juvenile hormone esterase gene expression by 20-hydroxyecdysone and juvenile hormone in Bombyx mori

Juvenile hormone esterase (JHE) is the primary juvenile hormone (JH) metabolic enzyme in insects and plays important roles in the regulation of molt and metamorphosis. We investigated its mRNA expression profiles and hormonal control in Bombyx mori larvae. JHE mRNA was expressed at the end of the 4th and 5th (last) larval instars in the midgut and in all the three (anterior, middle, posterior) parts of the silk gland. In the fat body, JHE expression peaked twice in the 5th instar, at wandering and before pupation, while it gradually decreased through the 4th instar. When 20-hydroxyecdysone (20E) was injected into mid-5th instar larvae, JHE mRNA expression was induced in the anterior silk gland but suppressed in the fat body. Topical application of a juvenile hormone analog fenoxycarb to early-5th instar larvae induced JHE expression in both tissues. In the anterior silk gland, JHE expression was accelerated and strengthened by 20E plus fenoxycarb treatments compared with 20E or fenoxycarb single treatment, indicating positive interaction of 20E and JH. JHE mRNA is thus expressed in tissue-specific manners under the control of ecdysteroids and JH.     

LncRNA63及其共表达基因Hr3在家蚕中的表达和功能分析

【目的】本研究旨在通过对一个新的长链非编码RNA (long non-coding RNA, lncRNA)及其共表达基因在家蚕Bombyx mori中的表达和功能进行分析,进一步阐明lncRNA在调控家蚕变态发育中的分子机制。【方法】基于前期家蚕脂肪体转录组测序数据,我们发现在蜕皮激素20E(2 μg/μL)处理5龄幼虫早期的脂肪体中lncRNA63表达显著上调,家蚕激素受体基因Hr3与之共表达。利用qRT-PCR对lncRNA63和Hr3在家蚕不同发育时期(4-5龄幼虫、蛹和成虫)、5龄幼虫不同组织(头、体壁、血淋巴、中肠、马氏管、脂肪体、丝腺、精巢和卵巢)和2 μg/μL 20E处理2, 6, 12和24 h后5龄幼虫脂肪体中的表达谱进行分析;采用核质分离检测和原位杂交技术检测lncRNA63在家蚕BmN细胞中的定位;利用dsRNA干涉家蚕5龄幼虫个体中lncRNA63的表达,并利用qRT-PCR检测RNA干涉lncRNA63对头、丝腺、脂肪体和体壁中Hr3表达的影响。【结果】qRT-PCR结果表明,lncRNA63和Hr3在家蚕羽化前的蛹末期有一个表达高峰;二者在家蚕5龄幼虫头、体壁和丝腺中都有较高表达;lncRNA63和Hr3在2 μg/μL 20E处理2, 6和12 h的家蚕5龄幼虫脂肪体中较对照组(无水乙醇处理组)都显著上调,24 h下降到与对照组相当水平,二者的表达趋势完全一致。LncRNA63主要定位于BmN细胞核,但20E可诱导lncRNA63的核质迁移。利用dsRNA干涉lncRNA63表达后,Hr3的表达显著下调。【结论】20E不仅可调控lncRNA63的表达,还可诱导lncRNA63的核质迁移;lncRNA63可通过调控共表达基因Hr3的表达参与家蚕的变态发育过程。     

家蚕BmlncR2036调控P25基因启动子活性及参与中肠发育调控

[目的]长链非编码RNA (long non-coding RNA,lncRNA)对家蚕Bombyx mori发育具有重要调控作用.我们在前期研究中发现一个位于家蚕丝素蛋白基因P25附近的lncRNA BmlncR2036.本研究旨在进一步探索BmlncR2036调控家蚕P25基因表达的分子机制.[方法]qPCR检测B...     

Translation of RNA from Eggs During Post-Diapause Development of Bombyx mori

Eggs of Bombyx mori are aroused from diapause by long-term chilling and develop when transferred to 25°C. During the first 20 hr of post-diapause development, the polysome content and the presumed rate of protein synthesis increase about 3-fold, while the ribosome content and the total RNA content increase only 1.1-fold. In this study, total RNAs were extracted from chilled eggs (termed 0 hr of development), and post-diapause eggs at 10 and 20 hr of development. The RNAs were purified further by high pressure liquid chromatography to remove RNA-like oligonucleotides. On translation in a protein-synthesizing system derived from wheat germ with a subsaturating amount of RNA, no difference was found in the relative amounts of translatable mRNA activity at 10 and 20 hr of development from that at 0 hr. Moreover, the translation products of the different RNA preparations in a rabbit reticulocyte lysate system appeared very similar when separated by gel electrophoresis and located by fluorography. These facts suggest that protein synthesis in early post-diapause development is controlled at a translational level.     

斜纹夜蛾线粒体复合物ⅢFe-S蛋白基因克隆、序列分析及在不同发育阶段的表达特征

Fe-S蛋白(rieske iron-sulfur protein, RISP)是线粒体复合物Ⅲ的关键蛋白亚基之一, 在呼吸链电子传递中起着重要作用。本文利用RT-PCR技术和RACE方法获得了斜纹夜蛾Spodoptera litura (Fab.)线粒体复合物ⅢRISP基因, 该基因序列全长4 413 bp, 含有3个内含子; 开放阅读框长816 bp, 编码271个氨基酸。经氨基酸序列比对, 与鳞翅目家蚕Bombyx mori和小菜蛾Plutella xylostella的Fe-S蛋白一致性较高, 分别为83%和79%, 与其他目昆虫的RISP一致性较低。qRT-PCR分析结果表明, SlitRISP基因在斜纹夜蛾不同发育阶段的表达水平存在显著差异, 即幼虫期和成虫期的表达量显著高于卵期和蛹期, 其中幼虫期又以第4和5龄幼虫表达水平显著高于初孵幼虫以及第1, 2, 3和6龄幼虫。斜纹夜蛾SlitRISP基因的克隆成功为今后对其功能研究以及作为靶标设计新型高效杀虫剂提供了基础。     

家蚕(Bombyx mori)末龄幼虫前胸腺内蜕皮素20-单氧酶活性(英文)

应用无细胞放射实验技术,逐日测定了家蚕末龄幼虫发育期间前胸腺和脂肪体匀浆液内蜕皮素20单氧酶(E-20-M)的活性。结果发现前胸腺内E-20-M活性特点是:从蜕皮到第3天幼虫前胸腺缺乏或具较低水平E-20-M活性;第4天前胸腺E-20-M活性开始升高;到第5天前胸腺E-20-M活性达到高峰。然而,与前胸腺相比,脂肪体E-20-M活性要高的多。     

苜蓿盲蝽气味结合蛋白基因Alin-OBP1的克隆及表达谱分析

有证据表明昆虫气味结合蛋白(odorant binding proteins, OBPs)与其嗅觉识别密切相关,起着运输外界脂溶性气味分子通过嗅觉感器淋巴液到达嗅觉受体的关键作用.为了更好地了解OBPs在苜蓿盲蝽Adelphocoris lineolatus (Goeze)嗅觉识别中的作用,本研究首次克隆了苜蓿盲蝽气味结合蛋白基因Alin-OBP1 (GenBank序列号GQ477022). 测序和序列分析结果表明,该基因开放阅读框长438 bp, 编码145个氨基酸,预测分子量为15.69 kDa,等电点为5.01,N-末端疏水区包含由18个氨基酸组成的信号肽.蛋白特征分析表明,该基因翻译后的蛋白质具有昆虫气味结合蛋白的典型特征,即氨基酸序列中有6个保守的半胱氨酸残基.利用RT-PCR和Real-time PCR技术对Alin OBP1在苜蓿盲蝽成虫不同组织和各个发育阶段的表达水平进行了测定,结果显示Alin-OBP1几乎全部在触角中表达.不同发育阶段Alin-OBP1表达量不同,在5龄若虫和成虫阶段表达水平最高.结果提示Alin-OBP1可能在苜蓿盲蝽感受包括性信息素在内的外界化合物的过程中发挥着重要作用.     

电穿孔法可用于家蚕基因功能分析

[目的]明确基于电穿孔的基因功能分析方法在家蚕Bombyx mori活体内的应用实效.[方法]针对调控家蚕幼虫体表斑纹黑色素合成的靶基因Wnt1(Wingless),人工合成特异性siRNA,向4龄第3天家蚕幼虫注射Wnt1 siRNA并进行电穿孔作为处理组(ERFA-RNAi),以注射Wnt1 siRNA但未进行电穿...