家蚕前部丝腺特异表皮蛋白Bm11721的鉴定及表达

家蚕的丝腺是其丝蛋白合成和分泌的器官,根据其形态和功能的不同分为前部、中部和后部丝腺,前部丝腺不具有合成丝蛋白的能力,是丝蛋白构象发生转变的场所。剪切力在丝蛋白构象转变中起到重要的作用,其在家蚕前部丝腺主要由前部丝腺逐渐变细的管腔结构和富含几丁质及表皮蛋白的坚硬的内壁提供。鉴定家蚕前部丝腺新的几丁质结合蛋白,并调查其在家蚕幼虫不同组织的表达特征。通过几丁质亲和层析的方法在前部丝腺筛选并鉴定到一个新的具有几丁质结合功能的表皮蛋白Bm11721,其编码基因编号为BGIBMGA011721(Gen Bank Accession No.NM-001173285.1)。利用原核表达系统成功表达了该蛋白,通过Ni-NTA亲和层析的方法获得了Bm11721的重组蛋白并制备了多克隆抗体。组织表达分析发现无论是转录水平还是蛋白水平Bm11721均只在前部丝腺特异表达,且Bm11721蛋白在5龄期的前部丝腺中恒定表达。免疫荧光定位结果显示Bm11721蛋白定位在前部丝腺的内膜中,推测其可能与前部丝腺的机械硬度有关,为丝蛋白的构象转变提供剪切力。     

Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx mori

Class B scavenger receptors (SR‐Bs) are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagocytosis of xenobiotics, and signaling. But little information is available about silkworm SR‐Bs; it is necessary to study these SR‐Bs for revealing their function. In this study, we cloned the full‐length coding sequence of BmSCRBQ4, a SR‐B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA (mRNA) was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue.     

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

Secretory Mechanism of Fibroin,a Silk Protein,in the Posterior Silk Gland Cells of Bombyx mori

There are two microtubule-microfilament systems in the posterior silk gland cells of Bombyx mori. One is a radial microtubule system; the other is a circular microtubule-microfilament system. These two systems are presumably concerned with the intra-cellular transport of secretory granules of fibroin and the secretion of fibroin into the lumen, respectively. Conventional and scanning electron microscopic observations of the two microtubule-microfilament systems in the posterior silk gland cells are reported. Scanning electron micrographs showed that a number of parallel linear cytoplasmic processes ran circularly on the luminal surface of the posterior silk gland cells. These processes were assumed to correspond to the circular microtubule-microfilament systems. The effects of cytochalasin (B or D), a secretion stimulating agent of fibroin, on the intracellular recording of membrane potential from the posterior silk gland cells are also reported. Exposure to cytochalasin resulted in depolarization of the membrane potential of the gland cells. Possible functional roles of the two microtubule-microfilament systems in the secretory mechanism of fibroin are discussed with reference to the effects of antimitotic reagents and cytochalasin on these two systems.     

Gene expression analysis in the larval silk gland of the eri silkworm Samia ricini

Insects produce silk for a range of purposes. In the Lepidoptera, silk is utilized as a material for cocoon production and serves to protect larvae from adverse environmental conditions or predators. Species in the Saturniidae family produce an especially wide variety of cocoons, for example, large, golden colored cocoons and those with many small holes. Although gene expression in the silk gland of the domestic silkworm (Bombyx mori L.) has been extensively studied, considerably fewer investigations have focused on members of the saturniid family. Here, we established expression sequence tags from the silk gland of the eri silkworm (Samia ricini), a saturniid species, and used these to analyze gene expression. Although we identified the fibroin heavy chain gene in the established library, genes for other major silk proteins, such as fibroin light chain and fibrohexamerin, were absent. This finding is consistent with previous reports that these latter proteins are lacking in saturniid silk. Recently, a series of fibrohexamerin‐like genes were identified in the Bombyx genome. We used this information to conduct a detailed analysis of the library established here. This analysis identified putative homologues of these genes. We also found several genes encoding small silk protein molecules that are also present in the silk of other Lepidoptera. Gene expression patterns were compared between eri and domestic silkworm, and both conserved and nonconserved expression patterns were identified for the tested genes. Such differential gene expression might be one of the major causes of the differences in silk properties between these species. We believe that our study can be of value as a basic catalogue for silk gland gene expression, which will yield to the further understanding of silk evolution.     

家蚕非编码RNA在神经系统中的表达分析

【目的】非编码RNA(non-coding RNA,ncRNA)在家蚕Bombyx mori发育过程中具有重要调控作用。本研究旨在探索ncRNA参与家蚕神经系统发育的分子机理。【方法】采用实时荧光定量PCR技术对22个中等长度的ncRNA及3个ncRNA的邻近编码基因在家蚕幼虫神经系统中的表达水平进行检测。【结果】8个ncRNA(包括1个C/D box snoRNA,4个H/ACA box snoRNA和3个不能归类的ncRNA)在家蚕5龄幼虫神经组织和非神经组织中均有表达,且在胸腹神经中的表达量明显高于头部神经中的表达量。其中,snoRNA Bm-51,Bm-18和Bm-86在胸腹神经中的表达量分别是头部神经中的23,5和4.7倍。进一步研究发现,这3个内含子起源的ncRNA与其宿主基因在家蚕神经系统中的表达趋势一致,宿主基因在胸腹神经中的表达量也明显高于头部神经中的表达量。【结论】本研究筛选到的在家蚕不同神经部位存在差异表达的ncRNA,特别是在胸腹神经中高表达的ncRNA可能协同其邻近编码基因,参与家蚕神经发育或神经活动过程。该结果为研究ncRNA参与家蚕神经系统发育提供了分子依据,为从非编码RNA角度探索鳞翅目害虫防治提供了新的思路。     

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BmlncR2036在5龄第3天家蚕幼虫不同组织(体壁、脑、神经、精巢、卵巢、丝腺、马氏管、血淋巴、脂肪体和中肠)中和不同发育阶段前丝腺、中丝腺和后丝腺中的表达谱。预测能够同时靶向P25和BmlncR2036的miRNA,利用荧光素酶检测法验证miRNA与P25互作关系。荧光素酶检测法测定BmlncR2036对P25基因启动子转录活性的影响。在家蚕5龄第3天幼虫中注射dsRNA敲低BmlncR2036的表达,观察丝腺及中肠表型的变化。【结果】qPCR结果显示,BmlncR2036在家蚕5龄第3天幼虫和5龄熟蚕的后丝腺中高表达,与P25基因呈现一致的表达趋势;BmlncR2036在家蚕5龄第3天幼虫的精巢和中肠中高表达,在其他组织中表达量较低,暗示其功能的多样性。miR-2739和miR-279a既能够与BmlncR2036匹配,也可能靶向P25基因的3′UTR。但荧光素酶检测法结果显示P25不是miR-2739和miR-279a的真实靶标。BmlncR2036负调控P25启动子活性,共转染pcDNA3.1(+)［BmlncR2036］和pGL3-Enhancer［P25-promoter］载体后,细胞荧光素酶活性极显著下降了52%。在家蚕5龄第3天幼虫中敲低BmlncR2036表达后出现体色变暗,中肠残留大量内容物,直至死亡的现象,表明BmlncR2036可能参与家蚕中肠的发育调控。【结论】发现lncRNA BmlncR2036可调控P25基因启动子活性,还可能参与调控家蚕中肠的发育。本研究为进一步探索家蚕lncRNA的功能提供了实验依据。     

Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori

Accumulating evidences show that small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. The silkworm is an important model for studies on insect genetics and control of lepidopterous pests. Here, we have performed the first systematic identification and analysis of intermediate size ncRNAs (50-500 nt) in the silkworm. We identified 189 novel ncRNAs, including 141 snoRNAs, six snRNAs, three tRNAs, one SRP and 38 unclassified ncRNAs. Forty ncRNAs showed significantly altered expression during silkworm development or across specific stage transitions. Genomic comparisons revealed that 123 of these ncRNAs are potentially silkworm-specific. Analysis of the genomic organization of the ncRNA loci showed that 32.62% of the novel snoRNA loci are intergenic, and that all the intronic snoRNAs follow the pattern of one-snoRNA-per-intron. Target site analysis predicted a total of 95 2'-O-methylation and pseudouridylation modification sites of rRNAs, snRNAs and tRNAs. Together, these findings provide new clues for future functional study of ncRNA during insect development and evolution.     

Ras1(CA) overexpression in the posterior silk gland improves silk yield

Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm, Bombyx mori, but has reached a plateau during the last decades. For the first time, we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%, while increasing food consumption by only 20%. Ras activation by Ras1(CA) overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins, up-regulated fibroin mRNA levels, increased total DNA content, and stimulated endoreplication. Moreover, Ras1 activation increased cell and nuclei sizes, enriched subcellular organelles related to protein synthesis, and stimulated ribosome biogenesis for mRNA translation. We conclude that Ras1 activation increases cell size and protein synthesis in the posterior silk gland, leading to silk yield improvement.