Identification and characterization of Polycomb group genes in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Polycomb group (PcG) proteins are involved in chromatin modifications for maintaining gene repression that play important roles in the regulation of gene expression, tumorigenesis, chromosome X-inactivation, and genomic imprinting in Drosophila melanogaster, mammals, and even plants. To characterize the orthologs of PcG genes in the silkworm, Bombyx mori, 13 candidates were identified from the updated silkworm genome sequence by using the fruit fly PcG genes as queries. Comparison of the silkworm PcG proteins with those from other insect species revealed that the insect PcG proteins shared high sequence similarity. High-level expressions of all the silkworm PcG genes were maintained through day 2 to day 7 of embryogenesis, and tissue microarray data on day 3 of the fifth instar larvae showed that their expression levels were relatively low in somatic tissues, except for Enhancer of zeste (E(Z)). In addition, knockdown of each PRC2 component, such as E(Z), Extra sex combs (ESC), and Suppressor of zeste 12 (SU(Z)12), considerably decreased the global levels of H3K27me3 but not of H3K27me2. Taken together, these results suggest that insect PcG proteins are highly conserved during evolution and might play similar roles in embryogenesis.     

Characterization of multiple <Emphasis Type="Italic">CYP9A</Emphasis> genes in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Based on the advances in the silkworm genome project, a new genome-wide analysis of cytochrome P450 genes was performed, focusing mainly on gene duplication. All four CYP9A subfamily members from the silkworm, Bombyx mori, were cloned by RT-PCR and designated CYP9A19–CYP9A22 by the P450 Nomenclature Committee. They each contain an open reading frame of 1,593 bp in length and encode a putative polypeptide of 531 amino acids. Both nucleic acid and amino acid sequences share very high identities with one another. The typical motifs of insect cytochrome P450, including the heme-binding region, helix-C, helix-I, helix-K, and PERF, show high sequence conservation among the multiple proteins. Alignment with their cDNA sequences revealed that these paralogues share identical gene structures, each comprising ten exons and nine introns of variable sizes. The locations of their introns (all nine introns follow the GT–AG rule) are absolutely conserved. CYP9A19, CYP9A20, and CYP9A21 form a tandem cluster on chromosome 17, whereas CYP9A22 is separated from the cluster by four tandem alcohol-dehydrogenase-like genes. Their phylogenetic relationships and structural comparisons indicated that these paralogues arose as the results of gene duplication events. RT-PCR detected their mRNAs in different “first line of defense” tissues, as well as in several other organs, suggesting diverse functions. Tissue-selective expression also indicates their functional divergence. The identified CYP9A genes have not yet been found outside the Lepidoptera, and are probably unique to the Lepidoptera. They show high sequence and structural similarities to each other, indicating that the Lepidoptera-specific P450s may be of functional importance. This analysis constitutes the first report of the clustering, spatial organization, and functional divergence of P450 in the silkworm.     

Alternative splicing of the <Emphasis Type="Italic">antitrypsin</Emphasis> gene in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Alternative splicing plays an important role in expanding protein diversity. In the present study, different splice variants of the antitrypsin gene (sw-AT) in the silkworm were identified by bioinformatics analyses using expressed sequence tags and genomic information. Four splice variants were obtained by RT-PCR with suitably designed primers, confirmed by sequencing, and designated as sw-AT-1, sw-AT-2, sw-AT-3, and sw-AT-4. The sw-AT gene contains 10 exons and nine introns. The splice variants differ in exon 9, with sw-AT-1, sw-AT-2, and sw-AT-3 using different versions of the exon, namely exon 9a, 9b, and 9c, respectively. In sw-AT-4, exon 9 consists of the combination of exons 9b and 9c. The expression patterns of the four isoforms in different tissues, at different developmental stages, and under different stress conditions (temperature, starvation, and mycotic infection) were characterized and quantified. The sw-AT isoforms showed tissue-specific expression patterns, with sw-AT-1 present in almost all tissues and sw-AT-4 found in only a few tissues. The four isoforms were predominantly expressed in the fat body, body wall, and testes of larvae, and exhibited similar expression profiles during development of the fat body. Among the stress treatments, low temperature had the greatest effect on isoform expression, and expression was also upregulated with mycotic infection.     

Genome-wide identification and evolution of <Emphasis Type="Italic">TC1</Emphasis>/<Emphasis Type="Italic">Mariner</Emphasis> in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>) genome

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated “cut and paste” mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.     

The genomic underpinnings of apoptosis in the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

Background

Apoptosis is regulated in an orderly fashion by a series of genes, and has a crucial role in important physiological processes such as growth development, immunological response and so on. Recently, substantial studies have been undertaken on apoptosis in model animals including humans, fruit flies, and the nematode. However, the lack of genomic data for silkworms limits their usefulness in apoptosis studies, despite the advantages of silkworm as a representative of Lepidoptera and an effective model system. Herein we have identified apoptosis-related genes in the silkworm Bombyx mori and compared them to those from insects, mammals, and nematodes.

Results

From the newly assembled genome databases, a genome-wide analysis of apoptosis-related genes in Bombyx mori was performed using both nucleotide and protein Blast searches. Fifty-two apoptosis-related candidate genes were identified, including five caspase family members, two tumor necrosis factor (TNF) superfamily members, one Bcl-2 family member, four baculovirus IAP (inhibitor of apoptosis) repeat (BIR) domain family members and 1 RHG (Reaper, Hid, Grim, and Sickle; Drosophila cell death activators) family member. Moreover, we identified a new caspase family member, BmCaspase-New, two splice variants of BmDronc, and Bm3585, a mammalian TNF superfamily member homolog. Twenty-three of these apoptosis-related genes were cloned and sequenced using cDNA templates isolated from BmE-SWU1 cells. Sequence analyses revealed that these genes could have key roles in apoptosis.

Conclusions

Bombyx mori possesses potential apoptosis-related genes. We hypothesized that the classic intrinsic and extrinsic apoptotic pathways potentially are active in Bombyx mori. These results lay the foundation for further apoptosis-related study in Bombyx mori.

     

Germ line transformation of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>, using the transposable element <Emphasis Type="Italic">Minos</Emphasis>

We investigated the use of Minos as a vector for transgenesis in the silkworm, Bombyx mori. We first constructed a vector plasmid with the green fluorescent protein (GFP) gene fused with the silkworm cytoplasmic actin gene (A3) promoter, and a helper plasmid with the Minos transposase gene controlled by the same A3 promoter. Injection of the vector and helper plasmid DNA into silkworm eggs produced transgenic animals in the following generation. The efficiency of transgenic silkworm production using this method was much lower than that obtained using piggyBac-mediated germ line transformation. However, >40-fold increase in the efficiency of producing transgenic silkworms was obtained using an in vitro synthesized source of Minos transposase mRNA. We conclude that the Minos transposon is a useful vector for construction of transgenic silkworms, particularly when in vitro synthesized mRNA is used. This is the first report showing that Minos can be used as a vector for germ-line transformation in lepidopteran insects.     

Comparative analysis of the genomes of <Emphasis Type="Italic">Bombyx mandarina</Emphasis> and <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedroviruses

The Bombyx mandarina nucleopolyhedrovirus (BomaNPV) S1 strain can infect the silkworm, Bombyx mori, but is significantly less virulent than B. mori nucleopolyhedrovirus (BmNPV) T3 strain. The complete nucleotide sequence of the S1 strain of BomaNPV was determined and compared with the BmNPV T3 strain. The circular, double stranded DNA genome of the S1 strain was 126,770 nucleotides long (GenBank accession no. FJ882854), with a G+C content of 40.23%. The genome contained 133 potential ORFs. Most of the putative proteins were more than 96% identical to homologs in the BmNPV T3 strain, except for bro-a, lef-12, bro-c, and bro-d. Compared with the BmNPV T3 strain, however, this genome did not encode the bro-b and bro-e genes. In addition, hr1 lacked two repeat units, while hr2L, hr2R, hr3, hr4L, hr4R, and hr5 were similar to the corresponding hrs in the T3 strain. The sequence strongly suggested that BomaNPV and BmNPV are variants with each other, and supported the idea that baculovirus strain heterogeneity may often be caused by variation in the hrs and bro genes.     

Molecular analysis of sex chromosome-linked mutants in the silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

In Bombyx mori, the W chromosome determines the female sex. A few W chromosome-linked mutations that cause masculinization of the female genitalia have been found. In female antennae of a recently isolated mutant, both female-type and male-type Bmdsx mRNAs were expressed, and BmOr1 (bombykol receptor) and BmOr3 (bombykal receptor), which are predominantly expressed in the antennae of male moths, were expressed about 50 times more abundantly in the antennae of mutant females than in those of normal females. These mutants are valuable resources for the molecular analysis of the sexdetermination system. Besides the Fem gene, the quantitative egg size-determining gene Esd is thought to be present on theW chromosome, based on the observation that ZWW triploid moths produce larger eggs than ZZW triploids. The most recently updated B. mori genome assembly comprises 20.5 Mb of Z chromosome sequence. Using these sequence data, responsible genes or candidate genes for four Z-linked mutants have been reported. The od (distinct oily) and spli (soft and pliable) are caused by mutation in BmBLOS2 and Bmacj6, respectively. Bmap is a candidate gene for Vg (vestigial). Similarly, Bmprm is a candidate gene for Md (muscle dystrophy), causing abnormal development of indirect flight muscle.     

Characterization and expression patterns of <Emphasis Type="Italic">let-7</Emphasis> microRNA in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>)

Background  

lin-4 and let-7, the two founding members of heterochronic microRNA genes, are firstly confirmed in Caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let-7 has been thought to trigger the onset of adulthood across animal phyla. Ecdysone and Broad-Complex are required for the temporal expression of let-7 in Drosophila melanogaster. For a better understanding of the conservation and functions of let-7, we seek to explore how it is expressed in the silkworm (Bombyx mori).     

Characterization of the <Emphasis Type="Italic">Bm61</Emphasis> of the <Emphasis Type="Italic">Bombyx mori</Emphasis> Nucleopolyhedrovirus

orf61 (bm61) of Bombyx mori Nucleopolyhedrovirus (BmNPV) is a highly conserved baculovirus gene, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this study, we describe the characterization of bm61. Quantitative polymerase chain reaction (qPCR) and western blot analysis demonstrated that bm61 was expressed as a late gene. Immunofluorescence analysis by confocal microscopy showed that BM61 protein was localized on nuclear membrane and in intranuclear ring zone of infected cells. Structure localization of the BM61 in BV and ODV by western analysis demonstrated that BM61 was the protein of both BV and ODV. In addition, our data indicated that BM61 was a late structure protein localized in nucleus.     

Identification and association of ISSR markers for thermal stress in polyvoltine silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

Evaluation of genetic resources is an essential prerequisite for their effective utilization. In India, the tropical climate prevails in most of the sericultural belts, where temperature goes beyond the ambient during summer, adversely affecting the silkworm rearing. Although polyvoltine silkworms are poor in silk content, they are mostly tolerant to tropical conditions and diseases. With an aim to identify potential silkworm races/breeds specific to thermotolerance for their effective utilization in breeding programme, 15 selected polyvoltine silkworm races were studied for their thermotolerance behaviour. Their genomic DNA samples were analyzed for ISSR-PCR using 15 selected primers. The UPGMA analysis based on Nei and Li algorithm has clustered the 15 silkworm races into five groups and one isolate. ALSCAL multidimensional scaling has not only supported the information generated by the dendrogram, but it has made the genetic distance among races more clear and substantiating their status in terms of thermal stress where pupation rate was taken as indicator. Further, discriminant function analysis (DFA) was done with three groups of silkworms classified for thermal stress viz. susceptible, moderately tolerant and tolerant. The canonical correlation value was estimated to be 0.987 (Wilk’s λ= 0.004; χ² = 36.044, p < 0.05). DFA clearly discriminated the above three groups. Beta statistics with t value and its significance for the markers identified through stepwise multiple regression analysis (MRA) revealed a total of five bands (807₁₃₀₀, 808₃₀₀₀, 808₄₀₀₀, 834₄₀₀₀, and 834₃₀₀₀) showing correlation with pupation rate after thermal treatment. Out of them, marker 808 3000 showed maximum and highly significant correlation (r = 0.757, p < 0.001, t = 4.182) with pupation rate among the silkworm races. The identified putative markers are being used to develop DNA marker to be used in evolving thermo-tolerant silkworm breeds using marker-assisted selection programme. The text was submitted by the authors in English.     

Molecular dissection of <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedrovirus <Emphasis Type="Italic">orf8</Emphasis> gene

Viruses including baculoviruses are obligatory parasites, as their genomes do not encode all the proteins required for replication. Therefore, viruses have evolved to exploit the behavior and the physiology of their hosts and often coevolved with their hosts over millions of years. Recent comparative analyses of complete genome sequences of baculoviruses revealed the patterns of gene acquisitions and losses that have occurred during baculovirus evolution. In addition, knowledge of virus genes has also provided understanding of the mechanism of baculovirus infection including replication, species-specific virulence and host range. The Bm8 gene of Bombyx mori nucleopolyhedrovirus (NPV) and its homologues are found only in group I NPV genomes. The Autographa californica NPV Ac16 gene is a homologue of Bm8 and, encodes a viral structural protein. It has been shown that Bm8/Ac16 interacts with baculoviral and cellular proteins. Bm8/Ac16 interacts with baculoviral IE1 that is facilitated by coiled coil domains, and the interaction with IE1 is important for Bm8 function. Ac16 also forms a complex with viral FP25 and cellular actin and associates with membranes via palmitoylation. These data suggested that this gene family encodes a multifunctional protein that accomplishes specific needs of group I NPVs.      

Novel non-autonomous transposable elements on W chromosome of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>

The sex chromosomes of the silkworm Bombyx mori are designated ZW(XY) for females and ZZ (XX) for males. Numerous long terminal repeat (LTR) and non-LTR retrotransposons, retroposons and DNA transposons have accumulated as strata on the W chromosome. However, there are nucleotide sequences that do not show the characteristics of typical transposable elements on the W chromosome. To analyse these uncharacterized nucleotide sequences on the W chromosome, we used whole-genome shotgun (WGS) data and assembled data that was obtained using male genome DNA. Through these analyses, we found that almost all of these uncharacterized sequences were non-autonomous transposable elements that do not fit into the conventional classification. It is notable that some of these transposable elements contained the Bombyx short interspersed element (Bm1) sequences in the elements. We designated them as secondary-Bm1 transposable elements (SBTEs). Because putative ancestral SBTE nucleotide sequences without Bm1 do not occur in the WGS data, we suggest that the Bm1 sequences of SBTEs are not carried on each element merely as a package but are components of each element. Therefore, we confirmed that SBTEs should be classified as a new group of transposable elements.