Xingfu Zha

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.

Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori

AbstractpiggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species.

Proteomic analysis of larval integument, trachea and adult scale from the silkworm, Bombyx mori

Multidimensional LC‐tandem MS was used to investigate the protein compositions of three tissues of silkworm, Bombyx mori. A total of 162, 259, and 175 peptides from silkworm larval integument and trachea, and adult scale obtained by database search were matched to 48, 51, and 40 proteins, respectively. Forty‐one cuticular proteins were identified from three tissues and covered all five cuticular protein families of silkworm. In the adult scale, all seven cuticular proteins were identified for the first time in the final pellet after SDS extraction. The majority of cuticular proteins were found in each tissue differentially, suggesting that tissue‐specific cuticular proteins were involved in the building of the specialized tissues. Seventy‐three non‐cuticular proteins were also identified in this analysis mainly including muscular proteins, proteinases, inhibitors, transport proteins, and redox‐related proteins.

Novel female-specific trans-spliced and alternative splice forms of dsx in the silkworm Bombyx mori.

The Bombyx mori doublesex gene (Bmdsx) plays an important role in somatic sexual development. Its pre-mRNA splices in a sex-specific manner to generate two female-specific and one male-specific splice forms. The present study investigated six novel dsx variants generated by trans-splicing between female dsx transcripts and two additional novel genes, dsr1 and dsr2. Expression analysis indicated that Bmdsx-dsr1 represented splicing noise, whereas dsr2, which trans-spliced with dsx to generate five variants, regulated the expression of the female-specific B. mori dsx transcript Bmdsx(F)s. We unexpectedly found a novel exon 2n insertion during Bmdsx transcription, which did not influence the validity of the novel protein, BmDSX(F3). Ectopic expression of BmDSX(F3) repressed the pheromone-binding protein gene and the testis-specific gene A2 in males, and activated of the storage protein 1 gene. Our findings suggest that trans-splicing is a novel regulatory function of Bmdsx, which participates in female sexual development by regulating the expression of three BmDSX(F) proteins.

Molecular cloning and expression analysis of Bmrbp1, the Bombyx mori homologue of the Drosophila gene rbp1

RBP1 is an important splicing factor involved in alternative splicing of the pre-mRNA of Drosophila sex-determining gene dsx. In this work, the Bombyx mori homologue of the rbp1 gene, Bmrbp1, was cloned. The pre-mRNA of Bmrbp1 gene is alternatively spliced to produce four mature mRNAs, named Bmrbp1-PA, Bmrbp1-PB, Bmrbp1-PC and Bmrbp1-PD, with nucleotide lengths of 799 nt, 1,316 nt, 894 nt and 724 nt, coding for 142 aa, 159 aa, 91 aa and 117 aa, respectively. BmRBP1-PA and BmRBP1-PD contain a N terminal RNA recognization motif (RRM) and a C terminal arginine/serine-rich domain, while BmRBP1-PB and BmRBP1-PC only share a RRM. Amino acid sequence alignments showed that BmRBP1 is conserved with its homologues in other insects and with other SR family proteins. The RT-PCR showed that Bmrbp1-PA was strongly expressed in all examined tissues and development stages, but Bmrbp1-PB was weakly expressed in these tissues and stages. The expression of both Bmrbp1-PA and Bmrbp1-PB showed no obvious sex difference. While the Bmrbp1-PC and Bmrbp1-PD were beyond detection by RT-PCR very likely due to their tissue/stage specificity. These results suggested that Bmrbp1 should be a member of SR family splicing factors, whether it is involved in the sex-specific splicing of Bmdsx pre-mRNA needs further research.

Cathepsin B protease is required for metamorphism in silkworm, Bombyx mori

Cathepsin B belongs to lysosomal cysteine protease of the papain family. Temporal and spatial expression analysis of cathepsin B of Bombyx mori (BmCtB) was carried out based on Expression Sequence Tags (ESTs) data, oligonucleotide microarray, reverse transcription polymerase chain reaction (RT‐PCR) and quantitative real‐time PCR. Expression of BmCtB was observed in all of the tissues and stages. Among the 10 tested tissues, the fat body and posterior silk gland are the two most enriched tissues with BmCtB. During Bombyx development, there was an expression fastigium of BmCtB during metamorphosis. RNA interference was used to suppress the expression of cathepsin B during metamorphosis. Significant developmental defective phenotypes were obtained in the RNAi treated group. The dramatically reduced expression of BmCtB was confirmed by Northern blot and quantitative real‐time PCR. These evidences strongly suggest cathepsin B protein‐ase was predominantly involved in the metabolism process of fat body and the posterior silk gland and was critical for metamorphism and development of silkworm, Bombyx mori.

Global expression profile of silkworm genes from larval to pupal stages： Toward a comprehensive understanding of sexual differences

Abstract  Sexual dimorphism is a widespread phenomenon in many higher animals. The genes and gene networks that underlie sex differences are poorly understood. Using microarray data we analyzed sex‐related differences in the global expression profiles of silkworm genes from larval to pupal stages. Sex‐biased genes could be divided into three clusters. Cluster 1 contained 932 genes that showed a female‐biased expression trend at first and a male‐biased trend afterward. Cluster 2 included 283 male‐biased genes. Cluster 3 was comprised of 497 female‐biased genes that were expressed during the late pupal stage. Cluster 1 genes were found to be related closely to cuticle proteins, hormones, binding proteins, enzyme regulators, structural proteins, transcription regulators and so on. Several genes in clusters 2 and 3 were associated with spermatogenesis and oogenesis, respectively. The chromosomal distribution of sex‐biased genes showed evidence of chromosomal enrichment. In particular a large number of the silkworms’ male‐biased genes are located on the Z chromosome. These results provide new insights into the molecular differences that dictate sexual dimorphism in the silkworm.

Species-specific expansion of C2H2 zinc-finger genes and their expression profiles in silkworm, Bombyx mori

Most C2H2 zinc-finger proteins (ZFPs) function as sequence-specific DNA-binding transcription factors, and play important roles in a variety of biology processes, such as development, differentiation, and tumor suppression. By searching the silkworm genome with a HMM model of C2H2 zinc-fingers, we have identified a total of 338 C2H2 ZFPs. Most of the ZFP genes were clustered on chromosomes and showed uneven distribution in the genome. Over one third of genes were concentrated on chromosome 11, 15 and 24. Phylogenetic analysis classified all silkworm C2H2 ZFPs into 75 families; 63 of which belong to evolutionarily conserved families. In addition, 188 C2H2 ZFP genes (55.6%) are species-specific to the silkworm. A species-specific expansion of a family with 39 members in a tandem array on chromosome 24 may explain the higher number of species-specific ZFPs in silkworm compared to other organisms. The expression patterns of C2H2 ZFP genes were also examined by microarray analysis. Most of these genes were actively expressed among different tissues on day 3 of the fifth instar. The results provide insight into the biological functions of the silkworm C2H2 ZFP genes in metamorphism and development.

Ectopic expression of the male BmDSX affects formation of the chitin plate in female Bombyx mori

Mating structures are involved in successful copulation, intromission, and/or insemination. These structures enable tight coupling between external genitalia of two sexes. During Bombyx mori copulation, the double harpagones in the external genitalia of males clasp the female chitin plate, which is derived from the larval eighth abdominal segment; abnormal development of the female chitin plate affects copulation. We report that ERK phosphorylation (p‐ERK) and expression of Abdominal‐B (Abd‐B) in the posterior abdomen of the female adult is lower than in the male. Ectopic expression of the male‐specific spliced form of B. mori doublesex (BmdsxM) in females, however, up‐regulates Abd‐B and spitz (spi) expression, increasing EGFR signaling activity, and thus forming an abnormal chitin plate and reduced female copulation. These findings indicate that Bmdsx affects the development of the eighth abdominal segment by regulating the activity of EGFR signaling and the expression of Abd‐B, resulting in an extra eighth abdominal segment (A8) in males versus the loss of this segment in adult females. Mol. Reprod. Dev. 2014.

New Insights into the Genomic Organization and Splicing of the Doublesex Gene, a Terminal Regulator of Sexual Differentiation in the Silkworm Bombyx mori

Sex-determination mechanisms differ among organisms. The primary mechanism is diverse, whereas the terminal regulator is relatively-conserved. We analyzed the transcripts of the Bombyx mori doublesex gene (Bmdsx), and reported novel results concerning the genomic organization and expression of Bmdsx. Bmdsx consists of nine exons and eight introns, of which two exons are novel and have not been reported previously. Bmdsx transcripts are spliced to generate seventeen alternatively-spliced forms and eleven putative trans-spliced variants. Thirteen of the alternatively-spliced forms and five of the putative trans-spliced forms are reported here for the first time. Sequence analysis predicts that ten female-specific, six male-specific splice forms and one splice form found in males and females will result in four female-specific, two male-specific Dsx proteins and one Dsx protein common to males and females. The Dsx proteins are expected to be functional and regulate downstream target genes. Some of the predicted Dsx proteins are described here for the first time. Therefore the expression of the dsx gene in B. mori results in a variety of cis- and trans-spliced transcripts and multiple Dsx proteins. These findings show that in B. mori there is a complicated pattern of dsx splicing, and that the regulation of splicing and sex-specific functions of lepidopteran dsx have evolved complexity.