Characterization of mitochondrial genome of Chinese wild mulberry silkworm, Bomyx mandarina (Lepidoptera: Bombycidae)

The complete mitochondrial genome of Chinese Bombyx mandarina(ChBm) was determined.The cir-cular genome is 15682 bp long, and contains a typical gene complement, order, and arrangement iden-tical to that of Bombyx mori(B.mori) and Japanese Bombyx mandarina(JaBm) except for two addi-tional tRNA-like structures:tRNASer(TGA)-like and tRNAIle(TAT)-like.All protein-coding sequences are initi-ated with a typical ATN codon except for the COI gene, which has a 4-bp TTAG putative initiator codon.Eleven of 13 protein-coding genes(PCGs) have a complete termination codon(all TAA), but the re-maining two genes terminate with incomplete codons.All tRNAs have the typical clover-leaf structures of mitochondrial tRNAs, with the exception of tRNASer(TGA)-like, with a four stem-and-loop structure.The length of the A T-rich region of ChBm is 484 bp, shorter than those of JaBm(747 bp) and B.mori(494―499 bp).Phylogenetic analysis among B.mori, ChBm, JaBm, and Antheraea pernyi(Anpe) showed that B.mori is more closely related to ChBm than JaBm.The earliest divergence time estimate for B.mori-ChBm and B.mori-JaBm is about 1.08±0.18―1.41±0.24 and 1.53±0.20―2.01±0.26 Mya, respec-tively.ChBm and JaBm diverged around 1.11±0.16―1.45±0.21 Mya.     

cDNA and deduced amino acid sequences of apolipophorin-IIIs from Bombyx mori and Bombyx mandarina

The cDNA sequence for apolipophorin-III from two strains of Bombyx mori (N4 and P50) and the Japanese and Chinese strains of Bombyx mandarina were determined. Both the cDNA and deduced amino acid sequences of the four apolipophorin-IIIs were highly similar (95-98%). The four Bombyx sequences also showed significant similarity to the sequence of apolipophorin-III from another lepidopteran, Manduca sexta (83-84%), particularly in the five amphipathic alpha-helices that are proposed to play a critical role in the binding of apolipophorin-III to lipophorin. In the coding region, the nucleotide sequences for the Chinese strain of B. mandarina and the P50 strain of B. mori were identical, supporting the suggestion that P50 is the current strain most closely related to the original domesticated strain. The N4 strain of B. mori is more closely related to these two strains than is the Japanese strain of B. mandarina, suggesting that Japanese strain of B. mandarina separated from the Chinese strain of B. mandarina before domestication of B. mori. Arch.     

野桑蚕、蓖麻蚕及家蚕基因组的RFLP分析

以家蚕Bombyx mori丝素重链基因、丝胶基因1和胰凝乳蛋白酶抑制因子13基因为探针,对野桑蚕B.mandarina、蓖麻蚕Philosamia cynthia ricini和家蚕B.mori基因组DNA进行限制性片段长度多态性分析。结果发现,在野桑蚕、蓖麻蚕基因组中存在着家蚕丝素重链基因、丝胶基因1的同源序列,而在中日野桑蚕以及蓖麻蚕品种间存在着限制性酶切位点差异;丝胶基因1在中国野桑蚕基因组的EcoRⅠ酶切图谱较日本野桑蚕与家蚕更为一致,表明家蚕与中国野桑蚕亲缘关系更近。此外,在野桑蚕基因组中发现了家蚕胰凝乳蛋白酶抑制因子13基因的同源序列,并且在家蚕品种间以及中日野桑蚕之间也存在着多态性。这些结果表明不同绢丝昆虫在适应生存环境的进化过程中,基因组发生了结构改变。     

Molecular phylogeny of the domesticated silkworm,Bombyx mori, based on the sequences of mitochondrialcytochrome b genes

Pupae from the Chinese wild mulberry silkworm,Bombyx mandarina, and 11 representative strains of the domesticated silkworm,Bombyx mori were selected for preparation of mitochondrial DNA. The 5′-end fragments ofcytochrome b genes (Cytb) were generated by polymerase chain reaction products and sequenced directly. The homologous sequences of the JapaneseB. mandarina and three strains ofB. mori were from the GenBank database. The sequences of the 16 silkworm strains were analysed with DNASTAR software and a phylogenic tree was constructed using PHYLIP software. The result showed that: (i) The sequence divergence between the strains ofB. mori and the JapaneseB. mandarina was larger (5.4% ≈ 5.8%) compared with that between strains ofB. mori and the ChineseB. mandarina (0.8% ≈ 1.9%). Analysis of clustering also showed that the sequences ofB. mori strains and ChineseB. mandarina clustered into group (B group), while that of JapaneseB. mandarina (A group) was outside this cluster. This may be evidence for the hypothesis thatB. mori originated from ChineseB. mandarina. (ii) Among 14 strains ofB. mori, sequence divergence was small and the most divergence was seen between strains Yanhe-1 and Chuxiong, whose sequences branched off from those of the otherB. mori strains on the phylogenetic tree. From this and from historical records, we infer that the strains Yanhe-1 and Chuxiong originated independently from southwest China.     

基于amy基因的中国野桑蚕遗传多样性及其与家蚕的系统发育关系

为探索中国野桑蚕Bombyx mandarina的遗传多样性及其与家蚕B. mori的系统发育关系, 采用PCR产物直接测序法（少数样本克隆测序）获得34个家蚕和野桑蚕样本淀粉酶基因amy序列片段（715 bp）。分析发现56个多态性位点, 鉴定出28种单倍型（haplotype）; 核苷酸多样性π=0.01390±0.00103, 单倍型多样度Hd=0.988±0.011。核苷酸不配对分析（mismatch analysis）和Fu’s Fs 检测表明中国野桑蚕曾发生过种群扩张。分子方差分析（AMOVA）表明, 遗野桑蚕传差异主要在种群内, 种群间和地理组群间差异不显著。聚类树上34个样本聚为3枝/3蔟, 野蚕和家蚕都不按地理区域或系统（类型）聚类, A枝由来自不同地区的野蚕和不同类型的家蚕混合构成, 并且进一步分成3个亚枝, 每一亚枝也同时包含家蚕和野蚕, B枝由3个家蚕和1个野蚕混合构成, C枝全部由来自不同地区的野蚕构成。网络分析没有发现“祖先单倍型”和优势单倍型。结果提示, 淀粉酶基因是一个多态性丰富的分子标记, 中国野桑蚕遗传多样性十分丰富, 据此推测家蚕起源于多种生态类型混杂的野桑蚕。     

Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA

Molecular phylogeny of some of the economically important silkmoths was derived using three mitochondrial genes, 12S rRNA, 16S rRNA, and COI, and the control region (CR). Maximum likelihood (ML) analyses showed two distinct clades, one consisting of moths from Bombycidae family and the other from Saturniidae family. The mitochondrial CR showed length polymorphisms with indels. The ML analyses for complete mitochondrial genome sequences of Bombyx mori (strains Aojuku, C108, Backokjam, and Xiafang), Japanese and Chinese strains of B. mandarina (Japanese mandarina and Chinese mandarina) and, Antheraea pernyi revealed two distinct clades, one comprising of B. mori strains and the other with B. mandarina, and A. pernyi forming an outgroup. Pairwise distances revealed that all of the strains of B. mori studied are closer to Chinese than to Japanese mandarina. Phylogenetic analyses based on whole mitochondrial genome sequences, the finding of a tandem triplication of a 126bp repeat element only in Japanese mandarina, and chromosome number variation in B. mandarina suggest that B. mori must have shared its recent common ancestor with Chinese mandarina. Another wild species of the Bombycidae family, Theophila religiosa, whose phylogenetic status was not clear, clustered together with the other bombycid moths in the study. Analysis of the interspecific hybrid, A. proylei gave evidence for paternal inheritance of mitochondrial DNA.     

野蚕黑卵蜂寄主识别利它素的纯化及氨基酸组成分析

针对利它素特殊的物理及化学性质 ,采用特定的温度分离方法 ,成功地从野蚕Theophilamandarina和家蚕Bombyxmori的雌蛾性附腺中分别获得了纯度较高的野蚕黑卵蜂Telenomustheophilae寄主识别利它素。对这两种利它素氨基酸组成的分析表明 ,来自家蚕和野蚕雌蛾性附腺的利它素在氨基酸的组成和含量方面非常相似 ,在检测到的 15种氨基酸中 ,甘氨酸、谷氨酸和天冬氨酸的克分子百分数在 10 %以上。从家蚕得到的分别为 2 8 1%、 18 5 %和 12 6 %。从野蚕得到的分别为 2 4 4%、18 1%和 10 1%。这 3种氨基酸之和在家蚕和野蚕中均超过 5 0 %以上。用凝胰乳蛋白酶对这两种利它素进行水解时发现溶液中均产生非水溶性沉淀 ,电泳表明沉淀的多肽蛋白分子量在 10kD左右。这显示在野蚕和家蚕利它素结构中 ,有着与家蚕丝蛋白相类似的结构 ,存在着结晶区域 (沉淀 )和无定形区域 (上清 )。     

M chromosome of the wild silkworm, Bombyx mandarina (n = 27), corresponds to two chromosomes in the domesticated silkworm, Bombyx mori (n = 28).

Chromosomes of Bombyx mori (n = 28) and of Bombyx mandarina (n = 27) were studied cytogenetically to resolve the origin of the large M chromosome in the Japaneses type of B. mandarina. In the F1 progeny from the reciprocal cross between B. mandarina and B. mori, the mitotic chromosome number was 2n = 55, and a chromosome configuration of 26 bivalents plus 1 trivalent was observed at metaphase I of germ cells. The trivalent chromosome consisted of the M chromosome from B. mandarina and two chromosomes from B. mori. When males of B. mori were mated to the F1 females, nuclei with two types of chromosome number (2n = 55 and 2n = 56) and two sets of chromosome pairs (26 bivalents plus 1 trivalent versus 28 bivalents) were observed in the metaphase I stage. Linkage analysis showed that the 14th chromosome of B. mori was involved in these two types of chromosome segregation. This result indicates that the M chromosome in B. mandarina arose from a fusion between a chromosome corresponding to the 14th linkage group and another, yet unidentified linkage group.     

中国野桑蚕遗传多样性的AFLP分析

采用AFLP技术对我国具有代表性的7个省市的10个野桑蚕(Bombyx mandarina)种群和2个家蚕（Bombyx mori)品种的遗传多样性进行了研究。结果表明：杭州、陕西和重庆3个地区的野桑蚕种群间及种群内个体间的遗传距离都比家蚕品种大。野桑蚕存在广泛的变异,7个省市的10个野桑蚕种群之间的遗传距离为0．164－0．444（平均值为0．3826）,平均杂合度为0．7061,表明野桑蚕自然群体的遗传多样性十分丰富。     

Anatomical and functional analysis of domestication effects on the olfactory system of the silkmoth Bombyx mori

The silkmoth Bombyx mori is the main producer of silk worldwide and has furthermore become a model organism in biological research, especially concerning chemical communication. However, the impact domestication might have had on the silkmoth''s olfactory sense has not yet been investigated. Here, we show that the pheromone detection system in B. mori males when compared with their wild ancestors Bombyx mandarina seems to have been preserved, while the perception of environmental odorants in both sexes of domesticated silkmoths has been degraded. In females, this physiological impairment was mirrored by a clear reduction in olfactory sensillum numbers. Neurophysiological experiments with hybrids between wild and domesticated silkmoths suggest that the female W sex chromosome, so far known to have the sole function of determining femaleness, might be involved in the detection of environmental odorants. Moreover, the coding of odorants in the brain, which is usually similar among closely related moths, differs strikingly between B. mori and B. mandarina females. These results indicate that domestication has had a strong impact on odour detection and processing in the olfactory model species B. mori.     

野桑蚕卵黄原蛋白的鉴定及cDNA序列分析

利用SDS-PAGE和Western blot方法分析鉴定了野桑蚕Bombyx mandarina Moore卵黄原蛋白,发现该蛋白由大小两个亚基组成,分子量分别为175 kD和42 kD。利用昆虫卵黄原蛋白进化上的保守性,根据家蚕的卵黄原蛋白cDNA序列设计特异性引物在野桑蚕的总RNA进行RT-PCR扩增,对于3′和5′端进行RACE扩增,解析获得了野桑蚕卵黄原蛋白cDNA全序列(GenBank登录号AY 309967)。该序列含有5.754个碱基,由一个开放阅读框组成,编码1.780个氨基酸,卵黄原蛋白的氨基酸序列与家蚕的同源性达到97.6%。在特定的酶切位点(RSRR)处,即第364～367个氨基酸位置,卵黄原蛋白前体被酶切为大小两个亚基,根据氨基酸推算的相对分子质量分别为161.571 kD和40.794 kD,如果考虑到翻译后的修饰,这与SDS-PAGE的结果是吻合的。同源性分析表明,昆虫卵黄原蛋白一级结构分化基本上局限在同一目内,具有较高的保守性。     

He—Ne激光辐照野桑蚕生物学效应研究

采用一定量He-Ne激光辐照野桑蚕催青卵及蚕肾,并与家蚕杂交,对其子代的蛋白质和酯酶同工酶进行电泳分析,结果发现谱带数目及活性有变化。另外,茧质调查和丝质调查与对照相比也有差异,并发现丝长和纤度特好的变异个体,显示He-Ne激光辐照对野桑蚕生理生化性状有一定影响。     

基于RAPD分析的中国野桑蚕和家蚕遗传多样性和系统发育关系研究

对具代表性的中国11个地区的野桑蚕Bombyx mandarina进行了随机引物扩增多态性DNA(RAPD)分析,结果表明：不同地区的野桑蚕的遗传距离较大,最大为0.465(安康-镇江),最小的也有0.209(武汉-合肥);同一地区不同个体野桑蚕的遗传距离也较大,最大为0.318,最小的为0.144。它们均远大于家蚕B.mori品种内个体间的遗传距离(最大为0.068、最小为0.015),甚至超过了家蚕品种间的遗传距离(最大为0.258、最小为0.197)。这表明中国野桑蚕是一个十分混杂的、遗传多样性非常丰富的群体。另外,在大多数情况下野桑蚕的遗传距离表现出与空间距离正相关。遗传距离和系统发育分析结果显示陕西一带的野桑蚕成分复杂,有重要的演化意义。     

野桑蚕酚氧化酶原基因cDNA的分子克隆及其特征

酚氧化酶在昆虫的免疫防御机制中起着重要作用。利用RT-PCR和RACE方法,克隆了野桑蚕酚氧化酶原基因,获得了其cDNA序列。该序列长2 134 bp,含有一个2 082 bp的完整开放阅读框,编码一个由693个氨基酸残基组成的蛋白质。推导的氨基酸序列与其他鳞翅目昆虫PPO2基因相应氨基酸序列有较高的同源性,该序列具有它们的PPO基因所共有的典型特征。组织特异性表达分析表明了该基因在野桑蚕5龄幼虫的血细胞、体壁、头部、精巢、卵巢、脂肪体和中肠等组织及其不同的发育阶段均有表达。这些结果为进一步研究野桑蚕酚氧化酶原基因的功能提供了分子基础。     

Specific expression of GFPuv-beta1,3-N-acetylglucosaminyltransferase 2 fusion protein in fat body of Bombyx mori silkworm larvae using signal peptide

Bombyxin (bx) and prophenoloxidase-activating enzyme (ppae) signal peptides from Bombyx mori, their modified signal peptides, and synthetic signal peptides were investigated for the secretion of GFP(uv)-beta1,3-N-acetylglucosaminyltransferase 2 (GGT2) fusion protein in B. mori Bm5 cells and silkworm larvae using cysteine protease deficient B. mori multiple nucleopolyhedrovirus (BmMNPV-CP(-)) and its bacmid. The secretion efficiencies of all signal peptides were 15-30% in Bm5 cells and 24-30% in silkworm larvae, while that of the +16 signal peptide was 0% in Bm5 cells and 1% in silkworm larvae. The fusion protein that contained the +16 signal peptide was expressed specifically in the endoplasmic reticulum (ER) and in the fractions of cell precipitations. Ninety-four percent of total intracellular beta1,3-N-acetylglucosaminyltransferase (beta3GnT) activity was detected in cell precipitations following the 600, 8000, and 114,000g centrifugations. In the case of the +38 signal peptide, 60% of total intracellular activity was detected in the supernatant from the 114,000g spin, and only 1% was found in the precipitate. Our results suggest that the +16 signal peptide might be situated in the transmembrane region and not cleaved by signal peptidase in silkworm or B. mori cells. Therefore, the fusion protein connected to the +16 signal peptide stayed in the fat body of silkworm larvae with biological function, and was not secreted extracellularly.     

野蚕黑卵蜂寄主识别利它素的纯化及氨基酸组成分析

针对利它素特殊的物理及化学性质,采用特定的温度分离方法,成功地从野蚕Theophila mandarina和家蚕Bombyx mori的雌蛾性附腺中分别获得了纯度较高的野蚕黑卵蜂Telenomus theophilae寄主识别利它素。对这两种利它素氨基酸组成的分析表明,来自家蚕和野蚕雌蛾性附腺的利它素在氨基酸的组成和含量方面非常相似,在检测到的15种氨基酸中,甘氨酸、谷氨酸和天冬氨酸的克分子百分数在10%以上。从家蚕得到的分别为28.1%、18.5%和12.6%。从野蚕得到的分别为24.4%、18.1%和10.1%。这3种氨基酸之和在家蚕和野蚕中均超过50%以上。用凝胰乳蛋白酶对这两种利它素进行水解时发现溶液中均产生非水溶性沉淀,电泳表明沉淀的多肽蛋白分子量在10 Kd左右。这显示在野蚕和家蚕利它素结构中,有着与家蚕丝蛋白相类似的结构,存在着结晶区域（沉淀）和无定形区域（上清）。     

Distinctive presence of peritracheal athrocytes in Bombyx mori L. and Bombyx mandarina M. as compared to their absence in several other lepidopteran species

Pericardial cells are present in a wide variety of insects and are thought to constitute the majority of 'athrocytes (nephrocytes)'. In contrast, peritracheal athrocytes have only been observed in Bombyx mori L. Although peritracheal athrocytes have a distinct morphology, it is unknown whether these cells are common to all lepidopterans. We anatomically compared eight lepidopteran species: Bombyx mori L. and Bombyx mandarina M. (Bombycidae); Samia cynthia ricini D. (Saturniidae); Agrius convolvuli L. (Sphingidae); Spodoptera litura F. and Mythimna separata W. (Noctuidae); Pieris rapae L. (Pieridae); and Glyphodes pyloalis W. (Crambidae). Of these species, only Bombyx mori L. and Bombyx mandarina M. possess peritracheal athrocytes.     

Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination

The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse.