家蚕免疫相关基因和信号途径的鉴定和比较分析

家蚕Bombyx mori是一种重要的经济昆虫, 在中国约有5 000年的驯化历史。家蚕分子免疫学方面的最新研究已经初步勾勒出其先天免疫的轮廓。本研究基于更新的家蚕基因组数据, 通过与黑腹果蝇Drosophila melanogaster、冈比亚按蚊Anopheles gambiae、意大利蜜蜂Apis mellifera和赤拟谷盗Tribolium castaneum基因组的比较分析, 鉴定了家蚕21个免疫相关基因家族的218个基因, 其编码产物包括模式识别受体、信号传导因子、效应分子和氧化防御相关的酶类。尽管信号传导因子的序列分化较大, 但系统进化分析显示它们在不同昆虫间呈明显的直系同源关系。相反, 与识别、调制和效应因子相关的基因的序列保守性更高, 但是这些基因家族明显缺乏直系同源基因, 由此推测这些基因是由物种特异的基因复制机制产生的。结果提示家蚕拥有与其他昆虫相同的免疫应答调控的分子机制, 而且家蚕同样可以通过基因复制及其序列分化等方式调节防御策略。     

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.     

家蚕Hox基因研究进展

Hox基因（homeobox genes）在昆虫躯体模式（body plan）的发育调控机制中扮演着重要角色,其表达具有严格的组织特异性和胚胎发育的程序性。家蚕Bombyx mori作为鳞翅目昆虫的代表,其Hox基因也陆续得到鉴定。在家蚕中存在一个拟复等位基因群--E群基因,其突变表型均与过剩斑纹和过剩附肢有关,这可能与Hox基因有着密切联系。家蚕全基因组测序完成后,发现其Hox基因簇中存在12个特有的homeobox基因（Bmshx1~Bmshx12）, 说明家蚕Hox基因可能具有独特的生物学意义。我们还利用家蚕基因芯片数据分析了Bmlab与Bmpb基因的组织表达特征。通过对家蚕Hox基因的研究,探索家蚕躯体模式建立机制,可望为解析其他鳞翅目昆虫的躯体模式的建立机制提供理论依据。本文就家蚕Hox基因的表达、功能及其与E群突变的关系等方面进行了综述。     

家蚕氨酰-tRNA合成酶基因分析

为了探讨家蚕氨酰-tRNA合成酶(BmaaRS)基因的数目、种类、结构及起源, 利用家蚕基因组数据和EST数据进行了BmaaRS基因的电子克隆, 结果表明, 家蚕核基因组中含有2套不同的aaRS核基因, 分别编码线粒体和细胞质BmaaRS, 但编码线粒体BmSerRS的基因有2个, 可能缺少编码细胞质的BmHisRS基因和编码线粒体的BmGlnRS、BmLysRS、BmGlyRS和BmThrRS基因, 这些基因的功能可能由具有相似功能的其他蛋白完成, 或通过某个BmaaRS基因的可变剪接分别形成不同功能的BmaaRS。EST证据表明, BmaaRS基因存在不同形式的可变剪接; BmaaRS氨基酸序列的相似性及二、三级结构分析表明部分BmaaRS存在结构域的扩增, 有些不同的BmaaRS具有相同结构域, 相同功能的BmaaRS具有相似的三级结构; 进化分析表明, BmaaRS为2套不同来源的BmaaRS基因编码, 细胞质和线粒体BmaaRS的起源不同。     

Systematic cloning and analysis of autophagy-related genes from the silkworm Bombyx mori

Background  

Through the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated.     

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.     

Dosage analysis of Z chromosome genes using microarray in silkworm,Bombyx mori

In many organisms, dosage compensation is needed to equalize sex-chromosome gene expression in males and females. Several genes on silkworm Z chromosome were previously detected to show a higher expression level in males and lacked dosage compensation. Whether silkworm lacks global dosage compensation still remains poorly known. Here, we analyzed male:female (M:F) ratios of expression of chromosome-wide Z-linked genes in the silkworm using microarray data. The expression levels of genes on Z chromosome in each tissue were significantly higher in males compared to females, which indicates no global dosage compensation in silkworm. Interestingly, we also found some genes with no bias (M:F ratio: 0.8–1.2) on the Z chromosome. Comparison of male-biased (M:F ratio more than 1.5) and unbiased genes indicated that the two sets of the genes have functional differences. Analysis of gene expression by sex showed that M:F ratios were, to some extent, associated with their expression levels. These results provide useful clues to further understanding roles of dosage of Z chromosome and some Z-linked sexual differences in silkworms.     

The chemistry of insect hemolymph; organic components of the hemolymph of the silkworm,Bombyx mori,and two other species

1. Hemolymph was collected for analysis from the silkworm, Bombyx mori, in a series of developmental stages ranging from the second molt to the late pupa. The mean pH of larval hemolymph after collection was found to be 6.45, that of pupal hemolymph, 6.57; in vivo values may be slightly lower. Total dry solids ranged from 5.4 to 10.6 per cent. Total protein ranged from 1.2 to 5.3 per cent, increasing rapidly during the fifth instar. 2. Free amino acids were separated chromatographically and estimated. Of 19 amino acids identified, amounting collectively to 823 to 1497 mg. per 100 ml., glutamine, histidine, and lysine generally occurred in greatest amount. Tryptophan was not detected, and cystine (or cysteine) was found in only one sample. The total free amino acids account for 35 to 55 per cent of the non-protein nitrogen of the plasma. 3. Free sugars, estimated semiquantitatively on chromatograms, comprise glucose, fructose, and sucrose in total amount ranging from about 5 to 40 mg. per 100 ml. Total acid-soluble, ultrafiltrable carbohydrate, estimated as glucose by the anthrone reaction, ranged from 166 to 635 mg. per 100 ml., indicating the presence of low molecular weight sugar derivatives. 4. Inorganic phosphate amounted to 5 to 15 mg. per 100 ml., and acid-soluble organic phosphate to 100 to 200 mg. per 100 ml. The latter fraction includes several substances, of which one was tentatively identified as glucose-6-phosphate and the remainder are as yet unidentified. 5. Single samples of hemolymph were also taken from larvae of the wax moth, Galleria mellonella, and the spruce sawfly, Diprion hercyniae. These contained even higher concentrations of solutes than the silkworm samples, but with a generally similar distribution. The proportions of the free amino acids were different in each species.     

不同家蚕品系的耐热性特征

研究了家蚕Bombyx mori 3个品系(Pure Mysore,NB4D2和CSR2）5龄幼虫和蛹在不同温度（35,38和40℃）下的耐热性,采用Probit分析测定了它们在各温度下的LT50值和置信限。结果表明：多化性品系Pure Mysore在高温下的存活率高于两个二化性品系NB4D2和CSR2,而两个二化性品系中NB4D2表现出更好的耐热性。家蚕幼虫接触38℃高温6 h和40℃高温3 h后,其血淋巴中出现90,70和29 kDa的热激蛋白条带。在恢复过程中,NB4D2和CSR2的血淋巴中未见29 kDa蛋白条带,而Pure Mysore幼虫的血淋巴中29 kDa蛋白仍然表达。当幼虫置于高温下时,血淋巴中90和70 kDa蛋白表达,但是检测不到29 kDa蛋白。研究认为热激蛋白表达与热带家蚕不同品系的耐热性以及与同一品系不同发育阶段的耐热性具有相关性。     

Bombyx small RNAs: genomic defense system against transposons in the silkworm, Bombyx mori

Selfish genetic elements called transposons can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Expansion of transposons can occur when novel transposition events are transmitted to subsequent generations after germline hopping. Therefore, organisms seem likely to have evolved defense mechanisms to silence transposons in the germline. Recently, small RNAs interacting with Piwi proteins (piwi-interacting RNAs: piRNAs) have been demonstrated to be involved in genomic defense mechanism against transposons. Here, we show that piRNA-like small RNAs are present abundantly in the Bombyx ovary. We cloned 38,493 kinds of Bombyx small RNA from the ovary and performed functional characterization. Bombyx small RNAs showed a unimodal length distribution with a peak at 28nt and a strong bias for U at the 5' end. We found that 12,869 kinds of Bombyx small RNAs were associated with transposons or repetitive sequences. We classified them as repeat-associated small interfering RNAs (rasiRNAs), a subclass of piRNAs. Notably, antisense rasiRNAs have a strong bias toward U at 5' ends; in contrast, sense rasiRNAs have a strong bias toward A at nucleotide position 10, indicating that the piRNA amplification loop proposed in Drosophila is evolutionarily conserved in Bombyx. These results suggest that Bombyx small RNAs regulate transposon activity.     

Starvation-responsive glycine-rich protein gene in the silkworm Bombyx mori

Four glycine-rich protein (GRP) genes were identified from expressed sequence tags of the maxillary galea of the silkworm. All four genes were expressed in the maxillary pulp, antenna, labrum, and labium, but none of the genes were expressed in most internal organs. Expression of one of the genes, termed bmSIGRP, was further increased approximately fivefold in the mouth region (including the maxilla, antenna, labrum, labium, and mandible) after 24 h of starvation. bmSIGRP expression peaked at 24 h and gradually declined during the subsequent 2 days. When a synthetic diet not containing proteins was fed, bmSIGRP expression increased significantly in the mouth region to levels similar to that observed in starved larvae. Synthetic diets that lacked vitamins or salts but contained amino acids did not significantly affect bmSIGRP expression. These results suggest that amino acid depletion increases bmSIGRP expression.     

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。