Identification and Characterization of 30 K Protein Genes Found in Bombyx mori (Lepidoptera: Bombycidae) Transcriptome.

The 30 K proteins, the major group of hemolymph proteins in the silkworm, Bombyx mori (Lepidoptera: Bombycidae), are structurally related with molecular masses of ∼30 kDa and are involved in various physiological processes, e.g., energy storage, embryonic development, and immune responses. For this report, known 30 K protein gene sequences were used as Blastn queries against sequences in the B. mori transcriptome (SilkTransDB). Twenty-nine cDNAs (Bm30K-1-29) were retrieved, including four being previously unidentified in the Lipoprotein_11 family. The genomic structures of the 29 genes were analyzed and they were mapped to their corresponding chromosomes. Furthermore, phylogenetic analysis revealed that the 29 genes encode three types of 30 K proteins. The members increased in each type is mainly a result of gene duplication with the appearance of each type preceding the differentiation of each species included in the tree. Real-Time Quantitative Polymerase Chain Reaction (Q-PCR) confirmed that the genes could be expressed, and that the three types have different temporal expression patterns. Proteins from the hemolymph was separated by SDS-PAGE, and those with molecular mass of ∼30 kDa were isolated and identified by mass spectrometry sequencing in combination with searches of various databases containing B. mori 30K protein sequences. Of the 34 proteins identified, 13 are members of the 30 K protein family, with one that had not been found in the SilkTransDB, although it had been found in the B. mori genome. Taken together, our results indicate that the 30 K protein family contains many members with various functions. Other methods will be required to find more members of the family.

Transcriptional regulation of the gene for prothoracicotropic hormone in the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) is one of key players in regulation of insect growth, molting, metamorphosis, diapause, and is expressed specifically in the two pairs of lateral PTTH-producing neurosecretory cells in the brain. Analysis of cis-regulatory elements of the PTTH promoter might elucidate the regulatory mechanism controlling PTTH expression. In this study, the PTTH gene promoter of Bombyx mori (Bom-PTTH) was cloned and sequenced. The cis-regulatory elements in Bom-PTTH gene promoter were predicted using Matinspector software, including myocyte-specific enhancer factor 2, pre-B-cell leukemia homeobox 1, TATA box, etc. Transient transfection assays using a series of fragments linked to the luciferase reporter gene indicated that the fragment spanning -110 to +33 bp of the Bom-PTTH promoter showed high ability to support reporter gene expression, but the region of +34 to +192 bp and -512 to -111 bp repressed the promoter activity in the BmN and Bm5 cell lines. Electrophoretic mobility shift assays demonstrated that the nuclear protein could specifically bind to the region spanning -124 to -6 bp of the Bom-PTTH promoter. Furthermore, we observed that the nuclear protein could specifically bind to the -59 to -30 bp region of the Bom-PTTH promoter. A classical TATA box, TATATAA, localized at positions -47 to -41 bp, which is a potential site for interaction with TATA box binding protein (TBP). Mutation of this TATA box resulted in no distinct binding band. Taken together, TATA box was involved in regulation of PTTH gene expression in B. mori.

Identification of a novel gene encoding the trehalose phosphate synthase in the cotton bollworm, Helicoverpa armigera.

Trehalose and trehalose metabolism are crucial for insect development. We measured the content of polyhydric compounds in the hemolymph of diapause- and nondiapause-destined individuals of Helicoverpa armigera. We found that the trehalose content is much higher in diapause-destined individuals than that in nondiapause individuals. The activity of trehalose-6-phosphate synthase (TPS) during H. armigera larval-pupal development is significantly higher in diapause-type individuals and is closely correlated with the changes in the trehalose content. The cDNA encoding TPS, which converts uridine-5'-diphosphoglucose and glucose-6-phosphate to trehalose-6-phosphate, was cloned from the fat body of H. armigera using rapid amplification of cDNA ends (RACE). The molecular characterization of the cDNA revealed that the mRNA encodes a precursor polypeptide of 826-amino-acid residues, containing Har-TPS at residues 6-507 and a putative trehalose-6-phosphate phosphatase, which converts trehalose-6-phosphate into free trehalose, at residues 512-783. The Har-TPS precursor polypeptide shows 73% identity with that of Drosophila melanogaster. The presence of a 2.8 kb transcript in the fat body and ovary was detected with a northern blot. The Har-TPS mRNA was detected at high levels in the late stage of sixth larval instar and the early middle stage of diapause-destined pupae, which are most likely to respond the changes in TPS activity and trehalose in the hemolymph. The Har-TPS protein was successfully overexpressed in the Bombyx mori baculovirus expression system, and the catalytic activity of Har-TPS was found to be approximately 5-fold higher in B. mori blood infected by the recombined-baculovirus than the control. When diapause is broken, the trehalose content drops significantly and glucose increases rapidly. These results suggest that trehalose is involved in regulating H. armigera pupal diapause.

Characterization of the Autographa californica nucleopolyhedrovirus ubiquitin gene promoter.

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes an ubiquitin protein, which may be involved in virus infection. Functional analysis of the AcMNPV ubiquitin promoter was performed by progressive deletion of sequence or mutation of putative cis-activating motifs in the promoter region. In the presence of viral factors, a transient expression assay demonstrated that the active regions responsive to promoter transcription are mainly located within the range of -595 to -382 bp upstream of ATG. A 196-bp fragment (-383 to -187 bp), consisting of the distal TAAG, CAAT motif and TATA box, could also drive the expression of a reporter gene. Site-directed mutagenesis analyses indicated that mutations of TATA boxes and TAAG motifs reduce the promoter activity remarkably, while CAAT mutations enhance the promoter activity by about 3- or 4-fold as compared to the native promoter. All the results suggested that two continuous promoter regions are involved in the transcription of the ubiquitin gene and the cis-activating motifs corresponding to viral factors are mainly present within the 5' region of the promoter. In addition, CAAT motifs in the promoter region function as negative regulator(s) binding sites.

Protein-DNA interactions in the promoter region of the gene encoding diapause hormone and pheromone biosynthesis activating neuropeptide of the cotton bollworm, Helicoverpa armigera.

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are two crucial neuropeptides which regulate insect development and sex pheromone biosynthesis respectively. These peptides are encoded by a single gene, termed DH-PBAN gene. In this study, we characterized the promoter of the DH-PBAN gene in Helicoverpa armigera (Har). Transient transfection assays using a series of stepwise deletion fragments linked to the luciferase reporter gene indicate that the promoter contains multiple regulator domains that can activate and repress reporter gene expression. The fragment spanning -467 to -371 bp of the DH-PBAN promoter is an activator domain of transcription, whereas the region from -965 to -534 bp represses the promoter activity in the insect cell line BmN. Electrophoretic mobility shift assays demonstrate that at least two nuclear protein factors from the nuclear protein extracts of H. armigera suboesophageal ganglion, Har-DHMBP-1 and-2 (DH-modulator-binding protein) can specifically bind to the activating region. Furthermore, we characterized in detail that the nuclear protein factor Har-DHMBP-3 can specifically bind to a classical E-box, CAGCTG localized at positions -360 to -355 bp, a potential site for interaction with basic helix-loop-helix transcription factors. Mutation of this E-box results in a significant reduction of the promoter activity, suggesting it can modulate the previously identified activator domain. Taken together, multipartite cis-elements and transcription factors in the DH-PBAN promoter are involved in regulation of the gene expression.

Characterization of CIb1 gene promoter from silkworm, Bombyx mori.

The hemolymph chymotrypsin inhibitor b1 (CIb1) of silkworm, Bombyx mori, plays an important role in innate immunity. In order to study its encoding gene CIb1, five heterogeneous promoter fragments of 844 bp, 682 bp, 516 bp, 312 bp and 82 bp in length were cloned from genomic DNA of the p50 silkworm strain. Characterization of the CIb1 promoter was performed in vitro using the firefly luciferase gene as reporter. The results showed that CIb1 promoter fragments have transcription activities in the B. mori ovary-derived BmN cell line. The 82 bp fragment (-72 to +10 nt) containing the eukaryotic core promoter elements revealed a basic transcription activity. The Bm1 element, upstream the transcription initiation site, showed a positive regulation function to the CIb1 promoter. CIb1 promoter-like fragments from the genomic DNA of the tetra hybrid silkworm SujuxMinghu provided a natural deletion model for the study of the CIb1 promoter. In vitro analysis indicated that the 132 bp fragment from -517 nt to -386 nt upstream of the transcription initiation site strongly suppressed the transcription activity of the CIb1 promoter, suggesting that the 132 bp fragment harbours strong negative cis-acting elements. Infection of Bombyx mori nucleopolyhedrovirus (BmNPV) increased the activity of the CIb1 promoter, having provided another evidence to the function of CIb1 in the innate immunity of silkworm.

Identification and analysis of YELLOW protein family genes in the silkworm, Bombyx mori.

BACKGROUND: The major royal jelly proteins/yellow (MRJP/YELLOW) family possesses several physiological and chemical functions in the development of Apis mellifera and Drosophila melanogaster. Each protein of the family has a conserved domain named MRJP. However, there is no report of MRJP/YELLOW family proteins in the Lepidoptera. RESULTS: Using the YELLOW protein sequence in Drosophila melanogaster to BLAST silkworm EST database, we found a gene family composed of seven members with a conserved MRJP domain each and named it YELLOW protein family of Bombyx mori. We completed the cDNA sequences with RACE method. The protein of each member possesses a MRJP domain and a putative cleavable signal peptide consisting of a hydrophobic sequence. In view of genetic evolution, the whole Bm YELLOW protein family composes a monophyletic group, which is distinctly separate from Drosophila melanogaster and Apis mellifera. We then showed the tissue expression profiles of Bm YELLOW protein family genes by RT-PCR. CONCLUSION: A Bombyx mori YELLOW protein family is found to be composed of at least seven members. The low homogeneity and unique pattern of gene expression by each member among the family ensure us to prophesy that the members of Bm YELLOW protein family would play some important physiological functions in silkworm development.

Functional Analysis of Helicase Gene Promoter and Homologous Region 3 Enhancer in Bombyx mori Nuclear Polyhedrosis Virus.

The promoter of the helicase gene, including 510 bp upstream of ATG,was cloned and sequenced, and was found that it had both early and late RNA initiation sites. The initiation codon ATG was deleted by using point mutation. Luciferase gene, as a reporter gene, was fused with the promoter region to construct the plsmid pBm hel 510 luc. When pBm hel 510 luc was transfected into Bm-5 and Sf-21 cell lines, the helicase gene promoter was recognized by cellular RNA polymerase and transactivated by viral factors. Baculovirus homologous regions (hrs) act as viral DNA replication start sites, which also have been shown to alter the rate of transcription for cis-linked promoters. BmNPV hr3 was cloned into a downstream site of luc gene, to study the effect of this enhancer on hel 510 promoter activity. The transient expression in transfected insect cell lines and silkworm larvae indicated that hr 3 could enhance the transcriptional level of hel 510 promoter by about 7 000 and 1 000 fold, respectively.

[Identification of functional region of helicase gene promoter in Bombyx mori nuclear polyhedrosis virus].

DNA helicases are essential for replication of baculoviruses. It was found that the helicase gene promoter of Bombyx mori nuclear polyhedrosis virus, including 510 bp upstream of ATG, had both early and late RNA initiation sites and could be recognized by cellular RNA polymerase. Transient expression assays in uninfected Sf-21 cells indicated that the helicase gene promoter could be classified as a delayed-early gene promoter. Deletion analysis by PCR showed that the regulation region of its basic transcription was mainly within -510 to -410 bp upstream of ATG. However, the basic activity was still detected with a deletion to -98 bp relative to ATG. In the presence of viral factors, deletion between -510 to -410 bp relative to ATG did not significantly reduce the promoter activity compared to the full-length promoter (510 bp). The remarkable reduction in the promoter activity was observed with continuous deletions. It suggests, therefore, that cis-acting elements responsive to viral factors are mainly located within the range of -410 to -309 bp upstream of ATG.

Promoter activities in the baculovirus envelope glycoprotein gp64 gene.

Baculovirus GP64 envelope glycoprotein is a specific major component of the envelope of the budded virus and is involved in virus entry into the host cells by endocytosis. For promoter activity analysis in the baculovirus gp64 gene, two DNA fragments containing 437 and 439 bp upstream of 5' ends of the BmNPV and AcMNPV gp64 ORF were amplified by polymerase chain reaction and cloned, respectively. The sequence analysis indicated that two gp64 genes have both early (CAGT) and late (A/GTAAG) transcriptional start sites. By use of the plasmids with a reporter luciferase gene (Luc) driven by gp64 promoter to transfect insect cells, transient expression assay showed that pBmgp64Luc had high expression levels in permissive Bm-N cells and very low levels in non-permissive Sf-21 cells, while pAcgp64Luc had relatively high expression levels both in permissive Sf-21 cells and in non-permissive Bm-N cells. Furthermore, the transcription of both gp64 promoters appeared to be transactivated by 2.4-4 times in corresponding permissive cells by corresponding viral factors, separately. By inserting BmNPV homologous region-3 (hr3) into the downstream of luciferase reporter gene driven by gp64 promoter, it enhanced transcription from both gp64 promoters by 13 - 22 times in Bm-N cells and over 7000 - 14,000 times in Sf-21 cells, respectively. In the presence of BmNPV hr3, correspondingly, the viral factors transactivated the transcriptional activity from two promoters by about 73 - 78 times in corresponding permissive cells. It suggested that BmNPV hr3 plays an important role in co-activation with viral factors onto the gp64 promoter besides the functions of viral DNA origin and enhancer.

The ecdysteroid UDP-glucosyltransferase gene promoter from Autographa californica multicapsid nucleopolyhedrovirus.

The ecdysteroid UDP-glucosyltransferase (egt) gene promoter fragments of different lengths were generated from the genomic DNA of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) by PCR. After being purified and enzymatic digestion, they were cloned into the pGEM-3Z vector for construction of reporter plasmids pAcegt542-luc, pAcegt309-luc and pAcegt159-luc with the luciferase gene driven by the AcMNPV egt promoter. The results of transient expression in the Spodoptera frugiperda cell line-21 (Sf21) showed that the transcriptional activity of the AcMNPV egt promoter required the transactivation of viral factor(s). The expression of luciferase gene driven by the AcMNPV egt promoter was first detected at 24 h post infection. The egt promoter from the Bombyx mori nucleopolyhedrovirus (BmNPV), closely related to AcMNPV, revealed similar properties to that of the AcMNPV egt promoter. When BmNPV homologous region 3 was subcloned downstream the luciferase gene, the luciferase activity of the reporter plasmid was enhanced by over 1000-fold, but the property of the promoter was not changed. As a substrate of ecdysteroid UDP-glucosyltransferase (EGT), foreign insect ecdysone showed negative effects on egt promoter transcriptional activity. Ecdysone of 1.0-2.0 microg/ml significantly down-regulated the promoter activity. Promoter activities of different lengths showed that an AcMNPV egt promoter fragment of 159 bp has the basal transcriptional activity but it was almost abolished only about 0.2% of that of 309 bp and 542 bp, respectively, and the nucleotide sequence from - 159 to - 309 nt upstream the translation initiation site includes the main cis-acting elements interacting with viral factors.

Proteomic analysis of the phenotype of the scaleless wings mutant in the silkworm, Bombyx mori.

A scaleless wing mutant of silkworm, Bombyx mori, has much fewer scales than wild type (WT). The scaleless phenotype was associated with tracheal system developmental deficiency and excessive apoptosis of scale cells. In this study, the wing discs proteins of WT and scaleless during pupation were studied using 2-DE and mass spectrometry. Of the 99 identified protein spots, four critical differentially expressed proteins between WT and scaleless were further verified using Q-PCR. At the first day of pupation (P0) in WT, imaginal disk growth factor (IDGF) was upregulated, whereas actin-depolymerizing factor 1 (ADF1) and profilin (PFN), which associated with cellular motility and cytoplasmic extension, were downregulated. We speculated their coaction counteracts the correct organization of the tracheal system in wing disc. Thiol peroxiredoxin (TPx) was upregulated in scaleless at P0, but its mRNA higher expression occurred in the day before pupation (S4). TPx could inhibit the formation of hydrogen peroxide, preventing the release of cytochrome C and activation of the caspase family protease. Its higher expression in scaleless was responsible for the apoptosis of scale cells delayed. The results provide further evidence that the scaleless phenotype was related to the tracheal system developmental deficiency and excessive apoptosis of scale cells.

The scaleless wings mutant in Bombyx mori is associated with a lack of scale precursor cell differentiation followed by excessive apoptosis.

The scaleless wings mutant in Bombyx mori (scaleless, sl) was previously reported morphologically. In the present study, we give data to clarify the mechanism of the mutation at the developmental level. Programmed cell death participates in the wing scale development during early pupal stage, and there are significant differences between that of sl and the wild type (WT) at each phase. Well-differentiated scale precursor cells do not form in sl when they have formed in WT. The peak of Caspase-3/7 activity in sl occurs 1 day later than and ten times as much as that in WT. Apoptotic bodies and DNA ladder studies also show that there is excessive apoptosis in sl early pupal wing. In addition, we have studied Bm-ASH1, an achaete-scute homolog in B.mori, which is thought to play a key role during the development of wing scales, and have found that the gene structure and expression levels of Bm-ASH1 in sl and WT are identical. All the data indicate that the wing scale precursor differentiation mechanism is abnormal in sl, which causes failing determination of scale cells and the downstream symptom of excessive apoptosis. But some of the elements to the scale differentiation circuit, such as Bm-ASH1, still operate in sl.