Structure and expression of the zebrafish mest gene, an ortholog of mammalian imprinted gene PEG1/MEST

PEG1/MEST is a paternally expressed gene in placental mammals. Here, we report identification of zebrafish (Danio rerio) gene mest, an ortholog of mammalian PEG1/MEST. Zebrafish mest encodes a polypeptide of 344 amino acids and shows a significant similarity to mammalian orthologs. Zebrafish mest is present as a single copy in the zebrafish genome and is closely linked to copg2 as in mammals. It is notable that 10 of 11 intron positions in mest are conserved among mammalian PEG1/MEST genes, indicating that the genomic organization and linkage between mest and copg2 loci was established in ancient vertebrates. Zebrafish mest is expressed in blastula, segmentation, and larval stages, exhibiting gradually increased expression as the development proceeds. Allelic expression analysis in hybrid larvae shows that both parental alleles are transcribed. We also observed one-codon alternative splicing involving an alternative usage of the two consecutive splice acceptors of intron 1, generating two protein isoforms with different lengths of a single amino acid.     

Differentially expressed genes in resistant and susceptible Bombyx mori strains infected with a densonucleosis virus

We investigated variations in the gene expression of Bombyx mori following infection with a densonucleosis virus (BmDNV-Z). Two B. mori near-isogenic lines, Jingsong and Jingsong.nsd-Z.NIL, which are highly susceptible and completely resistant to BmDNV-Z, respectively, were used in this study. The infection profiles of BmDNV-Z in the midguts of the B. mori Jingsong and Jingsong.nsd-Z.NIL larvae revealed that the virus invaded the midguts of both of these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, three cDNA libraries were constructed to compare BmDNV-Z responsive gene expression between the two silkworm lines. In total, 151 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that 11 genes were significantly up-regulated in the midgut of the Jingsong.nsd-Z.NIL strain following BmDNV-Z infection. Our results imply that these up-regulated genes might be involved in B. mori immune responses against BmDNV infection.     

家蚕瘫痪肽结合蛋白基因克隆与分析

ENF肽家族具有保守的N末端结构（Glu—Asn—Phe-）。该家族成员肽大多具有重叠功能活性,在鳞翅目昆虫的免疫反应,生长调控和自体调节等方面都发挥着重要的作用。在昆虫的免疫反应中,血细胞尤其是淋巴液的黏附性是针对外来侵入物的免疫应答过程中的重要因素。家蚕瘫痪肽（paralytic peptide）是ENF肽家族的一种,其具有多种的生物学活性,包括致瘫痪性及在家蚕血细胞免疫反应中的促吞噬细胞扩散活性。ENF肽家族的另一成员,粘虫（Pseudaletia separata）的生长阻抑肽（Growth-blocking peptide）,同家蚕瘫痪肽一样能够在粘虫的血细胞免疫反应中起到调节吞噬细胞的功能。目前,关于昆虫细胞免疫应答的终端调控分子机制的研究还比较少,有文献报道粘虫的生长阻抑肽结合蛋白（GBP—BP）能够起到沉默生长阻抑肽活性的功能,从而可能参与调节细胞免疫应答的终端调控。在本研究中,利用荧光差异显示技术（FDD）分析了家蚕感染BmNPV病毒后基因表达差异情况,在血淋巴中获得了一条差异条带G12782＊通过5＇-RACE技术,首次在家蚕中克隆得到了该基因的全长cDNA序列。通过同源性分析得知,该基因所编码的蛋白质与粘虫的生长阻抑肽结合蛋白具有很大的同源性,并被命名为家蚕瘫痪肽结合蛋白（Bmori paralytic peptide binding protein,PP-BP）。通过RT-PCR研究发现,该蛋白基因在血淋巴中大量表达。同时,利用实时荧光定量PCR（Real-time quantitative PCR）技术分析了该基因在正常饲养家蚕与添食BmNPV病毒的家蚕中的表达差异,结果显示该基因在家蚕添食BmNPV病毒后的表达量大大增强,这就暗示该基因可能与BmNPV病毒刺激后所引起的家蚕血液细胞免疫反应相关。利用生物信息学方法对该基因的结构进行了分析,发现该基因具有两个外显子和一个内含子。这个基因已经登入GenBank数据库,收入号为DQ306881。     

家蚕5龄幼虫精巢和卵巢microRNA芯片及转录组比较分析

【目的】本研究旨在通过对家蚕Bombyx mori 5龄幼虫精巢和卵巢组织微小RNA (microRNA, miRNA)基因芯片及转录组进行分析,找到参与家蚕性腺发育相关的miRNA分子及可能的靶基因。【方法】采用新一代高通量测序平台对家蚕5龄幼虫精巢和卵巢(分别定义为Test和Control)进行miRNA基因芯片检测及转录组测序分析,根据P＜0.05且log₂(fold change, FC)≥2的标准,通过比较筛选出Test vs Control的差异表达miRNA;根据q≤0.05且|log₂(fold change)|≥1的标准,通过比较筛选出Test vs Control的差异表达基因 (differentially expressed genes, DEGs);随机选取8个上调和12个下调差异表达miRNA,对其表达及其预测的5个靶基因进行qRT-PCR验证;对DEGs以及差异表达miRNA的靶基因进行KEGG通路富集分析。【结果】从精巢和卵巢样本中(Test vs Control)分别鉴定出68个差异表达miRNA和3 991个DEGs,其中上调和下调miRNA分别为36和32个,上调和下调DEGs分别为2 033和1 958个。差异表达miRNA的qRT PCR验证结果均与芯片数据一致。KEGG通路富集分析结果显示DEGs在新陈代谢及核糖体的信号通路显著富集。对差异表达miRNA在DEGs中的可能靶基因进行预测,结果找到了4组表达趋势相反的miRNA与靶基因：分别是bmo-miR-2774a与LOC101745556;bmo-miR-92b与LOC101735954以及bmo-miR-3266与LOC733130和LOC778467;1组表达趋势一致的miRNA与靶基因：bmo-miR-3321与LOC101744895。5个靶基因的qRT-PCR验证结果与转录组测序结果一致。【结论】本研究获得了家蚕5龄幼虫精巢和卵巢转录组及miRNA芯片数据,筛选并验证了4组差异表达和1组一致表达miRNA及潜在靶基因,为探究家蚕精巢和卵巢发育差异奠定了基础。     

Evidence of selection at melanin synthesis pathway loci during silkworm domestication

The domesticated silkworm (Bombyx mori) was domesticated from wild silkworm (Bombyx mandarina) more than 5,000 years ago. During domestication, body color between B. mandarina and B. mori changed dramatically. However, the molecular mechanism of the silkworm body color transition is not known. In the present study, we examined within- and between-species nucleotide diversity for eight silkworm melanin synthesis pathway genes, which play a key role in cuticular pigmentation of insects. Our results showed that the genetic diversity of B. mori was significantly lower than that of B. mandarina and 40.7% of the genetic diversity of wild silkworm was lost in domesticated silkworm. We also examined whether position effect exists among melanin synthesis pathway genes in B. mandarina and B. mori. We found that the upstream genes have significantly lower levels of genetic diversity than the downstream genes, supporting a functional constraint hypothesis (FCH) of metabolic pathway, that is, upstream enzymes are under greater selective constraint than downstream enzymes because upstream enzymes participate in biosynthesis of a number of metabolites. We also investigated whether some of the melanin synthesis pathway genes experienced selection during domestication. Neutrality test, coalescent simulation, as well as network and phylogenetic analyses showed that tyrosine hydroxylase (TH) gene was a domestication locus. Sequence analysis further suggested that a putative expression enhancer (Abd-B-binding site) in the intron of TH gene might be disrupted during domestication. TH is the rate-limiting enzyme of melanin synthesis pathway in insects. Real-time polymerase chain reaction assay did show that the relative expression levels of TH gene in B. mori were significantly lower than that in B. mandarina at three different developmental stages, which is consistent with light body color of domesticated silkworm relative to wild silkworm. Therefore, we speculated that expression change of TH gene may contribute to the body color transition from B. mandarina to B. mori. Our results emphasize the exceptional role of gene expression regulation in morphological transition of domesticated animals.     

对人工饲料摄食性不同的家蚕品种Cph2基因的表达特征分析

家蚕Bombyx mori不同品种对人工饲料的摄食性存在很大差异。为探讨食性差异的分子机理, 本文基于对人工饲料摄食性不同的蚕品种（系）SAGE （serial analysis of gene expression）文库差异表达基因的分析, 发掘了1条家蚕假定表皮蛋白（cuticular protein hypothetical）基因BmCph2。采用半定量RT-PCR和实时荧光定量PCR方法, 对BmCph2在不同摄食性蚕品种（系）不同发育时期的表达特征进行了研究。结果表明： BmCph2基因在家蚕幼虫眠期和起蚕期高表达, 在胚胎期和幼虫将眠期几乎检测不到表达; 在幼虫头部与全蚕的表达特征相似, 而在中肠中表达活性很低, 推测该基因表达可能与家蚕新表皮的形成有关。BmCph2在对人工饲料摄食性不同的蚕品种（系）中的表达存在较大差异, 在摄食性好的高食性品种中表达量显著低于摄食性差的低食性品种; 饲料和忌避剂的气味刺激及取食刺激对不同品种（系）该基因的表达有不同的影响, 高食性蚕对诱导刺激比较敏感, 而低食性蚕受影响较小, 尤其是菁松A和菁松B的低食性品系几乎不受影响。本研究结果说明, BmCph2基因除可能参与表皮形成的同时, 还与家蚕的食性有密切关系, 但其具体机理有待于进一步研究。     

Gene expression profiling between embryonic and larval stages of the silkworm, Bombyx mori

To elucidate the molecular mechanisms associated with metamorphic phenomenon relating to Bombyx mori, an important organism in the sericulture industry, we identified genes that are expressed in the different developmental stages, specifically the embryonic (ES) and larval (LS) stages of B. mori. Of 8230 high-quality ESTs from two full-length enriched cDNA libraries, 3442 of the ES ESTs were coalesced into 1325 clusters, while 4788 were coalesced into 927 clusters. The functional classification of these ESTs based on Gene Ontology showed that the types of genes that are associated with oxidoreductase activity, enzyme inhibition, and larval development were highly observed in LS, whereas the types of genes that are involved in nucleotide binding, enzyme activity, and protein transport activity were highly observed in ES. In addition, when the gene expression profile between ES and LS was examined by counting the EST frequencies in each library, 69 genes were identified as being either up- or down-regulated in the larval stage compared to the embryonic stage (P>0.99) and this was confirmed by semi-quantitative RT-PCR. The results show that genes involved in proteolysis and peptidolysis, and lipid and carbohydrate metabolism were dramatically up-regulated in LS, while those related to protein metabolism, DNA/RNA, and coenzymes were highly down-expressed. In particular, a GO analysis of these genes revealed that genes that are involved in hydrolase activity were observed to be highly expressed in amount as well as diversity in LS, while those involved in nucleic acid binding were highly expressed in ES. These data may contribute to elucidating genetic events that distinguish the developmental stage and to our understanding of the metamorphosis of B. mori.     

Genome-wide analysis of cytochrome P450 monooxygenase genes in the silkworm, Bombyx mori

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed. A total of 84 CYP-related sequences were identified and could be classified into 26 families and 47 subfamilies according to standard nomenclature. Seventy eight of the eighty four genes appear to be functional and six are probable pseudogenes. The distribution of Bombyx mori P450s in the genome shows that most of them are tandem arranged on chromosomes, only 34 genes are present as singletons, with 8 clusters including 3 or more than 3 genes. Sequence alignments were used to reconstruct phylogenetic trees and to analyze the intron-exon organizations of the functional genes. The conserved intron positioning agrees perfectly with their common grouping on the tree. The presence of three extremely ancient introns which are conserved across different clans indicates that a few introns are still highly conserved after they have undergone extensive evolutionary changes of B. mori P450 duplication and divergence. Comparison of the P450s from B. mori to the P450s from Drosophila melanogaster shows that the expansion is not uniform across the gene families. Remarkably, two mitochondrial families, the B. mori CYP333 and D. melanogaster Cyp12, formed two orthologous groups in the phylogenetic tree. All CYP333s can be proposed to be related to xenobiotic metabolism in accordance with the D. melanogaster Cyp12s. The characterization and evolutionary analysis of P450s from B. mori in the current study provide useful information for understanding the characteristics and diversity of P450s from B. mori and the baseline for functional analyses of individual P450s in this model Lepidopteran insect.     

家蚕生殖细胞相关基因nanos的克隆表达及在胚胎发育中的表达模式

【目的】探讨nanos基因在家蚕Bombyx mori胚胎发育中的表达模式,为进一步研究该基因在家蚕胚胎发育中的功能奠定基础。【方法】根据本实验室提交到GenBank中家蚕nanos的cDNA序列(登录号EF647589)设计引物,扩增出了一条长684 bp的编码片段,对该片段进行了克隆和表达,亲和纯化表达的蛋白并免疫新西兰大白兔制备抗体。Western blot检测家蚕早期胚胎nanos的表达情况,荧光定量PCR检测nanos在整个家蚕胚胎发育中的表达情况。【结果】克隆并表达了一条长684 bp的编码片段,得到了分子量约33 kD的融合蛋白。用制备的抗血清对家蚕早期胚胎蛋白的Western blot检测表明,nanos在此阶段基本是恒定表达。荧光定量PCR结果显示刚产的卵中nanos的表达量最大,第2天开始急剧下降,此后到第10天表达量几乎没有变化。【结论】本实验克隆的nanos是家蚕中的一个同源物,该基因在家蚕胚胎发育中的表达模式与蜜蜂等有很大的不同,反映了昆虫生殖细胞形成机制的多样性。     

Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori

To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1-4, in the Bombyx genome. Northern blot and RT-PCR analysis showed that BmGlov1-4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-kappaB binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1-4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1-4 significantly inhibited the growth of E. coli.