Disappearance of chorion proteins from Bombyx mori eggs treated with HCl solution to prevent diapause

Bombyx mori eggs enter diapause immediately after completion of mesoderm segregation. HCl treatment of approximately 24-hour-old eggs (germband formation stage) is well known to be the most effective procedure to prevent entry into diapause, although the molecular mechanism remains unclear. In this study, we examined the protein composition of diapausing and nondiapausing eggs after various HCl treatments known to prevent or break diapause and found that proteins of approximately 11 and 8 kDa disappeared immediately after HCl treatment. Partial amino acid sequences of these proteins indicated that they were members of the chorion class A protein L12 family synthesized in follicle cells. Under the hypothesis that the chorion provides a barrier to oxygen, dechorionation of diapausing eggs induces resumption of embryonic development. Hence, to test this and other hypotheses about the function of these proteins, we used 20% SDS-PAGE with Coomassie Brilliant Blue staining to trace their disappearance from embryos and eggshells after treatment with HCl under different conditions and on polyvoltine, univoltine, and bivoltine silkworm races. Even when 10-day-old diapausing eggs were treated with HCl, which did not break diapause, the 11 and 8 kDa proteins disappeared. Our results suggest that disappearance of these proteins is not directly associated with preventing entry into or breaking a diapause state. Nevertheless, our results cannot completely rule out the possibility that the 11 and 8 kDa proteins function to block permeability of O₂ during the period when HCl treatment is physiologically effective to prevent diapause so that after the diapause system is established within the egg, even removing the 11 and 8 kDa proteins may not affect to prevent diapause. We also discuss the role of these proteins in choriogenesis.     

Overexpression and functional characterization of an Aspergillus niger phytase in the fat body of transgenic silkworm,Bombyx mori

In a previous study, we isolated 1,119 bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30 K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5–2.0 and 5.5–6.0), and two optimum temperatures (55 and 37 °C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84 % of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms.     

The promoter of Bmlp3 gene can direct fat body-specific expression in the transgenic silkworm, Bombyx mori

The fat body plays multiple, crucial roles in the life of silkworms. Targeted expression of transgenes in the fat body of the silkworm, Bombyx mori, is important not only for clarifying the function of endogenous genes expressed in this tissue, but also for producing valuable recombinant proteins. However, fat body-specific gene expression remains difficult due to a lack of suitable tissue-specific promoters. Here we report the isolation of the fat body-specific promoter of Bmlp3, a member of the 30K protein family of silkworms. The 1.1 kb fragment from ?374 to +738 of Bmlp3 displayed strong promoter activity in the cell lines BmE and Spli-221. In transgenic silkworms, a DsRed reporter gene controlled by the 1.1 kb Bmlp3 promoter fragment was expressed specifically in the fat body in a stage-specific pattern that was nearly identical to the endogenous Bmlp3 gene. We conclude that the 1.1 kb Bmlp3 promoter fragment is sufficient to direct tissue- and stage-specific expression of transgenes in the fat body of silkworms, highlighting the potential use of this promoter for both functional genomics research and biotechnology applications.     

Genetic structure,polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella

Laboratory of genetics and physiology 2 (LGP2) is an actual detector and regulator during RNA viral infection in innate immunity. In this study, 5′-flanking region and all introns of LGP2 in grass carp (Ctenopharyngodon idella) were excavated. The genomic CiLGP2 (C. idella LGP2) was 8062 bp in length, with a 364 bp 5′-flanking region, twelve exons and eleven introns. Besides, the promoter activity of the upstream region before initiator codon was identified. By sequencing, six single nucleotide polymorphisms (SNPs) and one 20-bp insertion/deletion polymorphism were detected in CiLGP2. With a challenge experiment, the genotype and allele distributions of these seven polymorphisms were examined. Analytic result revealed only the − 1392 C/G, 494 A/T and 4403 C/T loci were significantly associated with the resistance/susceptibility to grass carp reovirus (GCRV) (P < 0.05). To further identify these correlations, another independent challenge test was performed. The analytic result based on the cumulative mortality demonstrated that the stock in − 1392 GG genotype was more susceptible to GCRV than that in CC genotype, while the stocks in 494 TT genotype and 4403 TT genotype were more resistant to GCRV than that in AA and CC genotype stocks, respectively (P < 0.05). Those significant SNPs might be potential gene markers for the future molecular selection of C. idella strains that are resistant to GCRV.     

Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.     

Nile Tilapia Neu3 sialidases: Molecular cloning,functional characterization and expression in Oreochromis niloticus

Mammalian Neu3 is a ganglioside specific sialidase. Gangliosides are involved in various physiological events such as cell growth, differentiation and diseases. Significance of Neu3 and gangliosides is still unclear in aquaculture fish species. To gain more insights of fish Neu3 sialidases, molecular cloning and characterization were carried out in tilapia (Oreochromis niloticus). A tilapia genome-wide search for orthologues of human NEU1, NEU2, NEU3 and NEU4 yielded eight putative tilapia sialidases, five of which were neu3-like and designated as neu3a, neu3b, neu3c, neu3d and neu3e. Among five neu3 genes, neu3a, neu3d and neu3e were amplified by PCR from adult fish brain cDNA with consensus sequences of 1227 bp, 1194 bp and 1155 bp, respectively. Multiple alignments showed conserved three Asp-boxes (SXDXGXTW), YRIP and VGPG motifs. The molecular weights for Neu3a, Neu3d and Neu3e were confirmed using immunoblotting analysis as 45.9 kDa, 44.4 kDa and 43.6 kDa, respectively. Lysate from neu3 genes transfected HEK293 cells showed sialidase activity in Neu3a towards ganglioside mix optimally at pH 4.6. Using pure gangliosides as substrates, highest sialidase activity for Neu3a was observed towards GD3 followed by GD1a and GM3, but not GM1. On the other hand, sialidase activities were not observed in Neu3d and Neu3e towards various sialoglycoconjugates. Indirect immunofluorescence showed that tilapia Neu3a and Neu3d are localized at the plasma membrane, while most Neu3e showed a cytosolic localization. RT-PCR analyses for neu3a showed significant expression in the brain, liver, and spleen tissues, while neu3d and neu3e showed different expression patterns. Based on these results, tilapia Neu3 exploration is an important step towards full understanding of a more comprehensive picture of Neu3 sub-family of proteins in fish.     

Fat body catalase activity as a biochemical index for the recognition of thermotolerant breeds of mulberry silkworm, Bombyx mori L.

The fifth instar larvae of the silkworm Bombyx mori L. were exposed to selected high temperatures (35 and 40 °C) in order to understand the changes in the level of catalase activity in the three tissues of fat body, midgut and haemolymph of the five selected bivoltine breeds and their 9 quantitative traits, namely larval weight, cocoon weight, shell weight, shell ratio, filament length, filament weight, denier, renditta and effective rearing rate (ERR), also the correlation between them under high temperature conditions were examined. Catalase activity resulting in fat body revealed a positive correlation between the control (28±1 °C) and 40±1 °C. The CSR₂ breed showed the most level of thermotolerance and catalase activity, compared with the CSR₄, JROP, NB₄D₂ and KA breeds. It was found that the level of catalase activity in fat body may be a reliable biochemical index to recognize thermotolerant breeds in order to develop resistant hybrids for tropical areas.     

Establishment and characterization of the Bombyx mandarina cell line

A new cell line, designated as NIAS-Boma-529b, was established from the larval fat bodies of Bombyx mandarina (B. mandarina), which is believed to be an ancestor of Bombyx mori (B. mori). This cell line has been cultured for approximately 150 passages during 2 years in an IPL-41 medium supplemented with 10% fetal bovine serum at a constant temperature of 26 °C. The morphology of this line includes adhesive round and spindle-shaped cells. Random-amplified polymorphic DNA analysis (RAPD) using 7 primers and a statistical analysis based on Nei’s genetic distance revealed that this cell line was closely related to B. mori-derived cell lines. An infection study also revealed that this cell line was susceptible to B. mori nucleopolyhedrovirus (BmNPV); however, it had no apparent susceptibility to Autographa californica NPV (AcNPV), which is closely related to BmNPV. Nevertheless, cells infected with AcNPV showed an extensive cytopathic effect (CPE), including a rough cell surface, rounding, nuclear expansion, and cell blebbing. These results suggest that this cell line can be useful to clarify the mechanism of host range determination of BmNPV and AcNPV.     

Expression of the hGM-CSF in the silk glands of germline of gene-targeted silkworm

To express human granulocyte-macrophage colony-stimulating factor (hGM-CSF) gene in the silk glands of transformation silkworm (Bombyx mori) based on gene-targeting, two fragments from fibroin heavy chain gene (fib-H) of silkworm were cloned and sequenced. One fragment contains the 1st exon and its downstream 1st intron’s partial sequence; and the other fragment contains the 1st intron’s partial sequence and the 2nd exon’s partial sequence. Then the two fragments, as homologous arm, were inserted into pSK to generate a gene-targeted vector, pSK-HL-A3GFP-FLP-GM-CSF-FLPA-HR in which a gfp gene driven by A3 promoter and an hGM-CSF gene under the control of fibroin light chain (fib-L) promoter were included. The vector was transferred into the silkworm eggs using sperm-mediated gene transfer. After being screened for green fluorescent, the transformation silkworm was obtained, whose genome was verified by PCR and dot hybridization to confirm whether the target genes had been integrated into the silkworm genome. Furthermore, in the posterior silk glands of the G4 generation transformation silkworms, a specific band with the molecular weight of 22 kDa could be detected by Western blotting with an antibody against hGM-CSF, and the expression level of the hGM-CSF estimated by ELISA was approximately 1.26 ng per gram fresh posterior silk gland.     

Characterization of Beauveria bassiana neutral trehalase (BbNTH1) and recognition of crucial stress-responsive elements to control its expression in response to multiple stresses

A neutral trehalase (NTH1) of fungal entomopathogen Beauveria bassiana was characterized for the first time as a 743-aa enzyme (84.4 kDa). To identify crucial stress-responsive elements (STREs) to control the expression of the NTH-coding gene (BbNTH1) in response to different stresses, the full-length promoter (−2713 bp) upstream of its open reading frame and three upstream-truncated fragments (−1912, −1060 and −560 bp) were fused to the reporter gene eGFP and then transformed into B. bassiana, respectively. Consequently, eGFP was well expressed as intensive fluorescence in mycelia, conidiogenic cells and forming conidia controlled by the full-length promoter with five STREs. Surprisingly, transformants controlled by the shortest fragment with last two STREs at −315 and −274 bp exhibited consistently brightest fluorescence in mycelia under 3-h oxidative adaption of 0.3-1.2 mM menadione, and in colonies under 6-day osmotic stress of 0.5-1 M NaCl and thermal stress of 15-540 min at 40 °C after 3-day growth at 25 °C. Single or dual site-directed mutations of the two STREs from CCCCT to CATCT significantly altered the gene response to the multiple stresses. Thus, the two STREs in the downstream 560-bp region of the promoter are crucial to regulating not only constitutive but stress-inducible expression of the target gene.