Toxicological assessment of pollen from different bt rice lines on Bombyx mori (Lepidoptera: Bombyxidae).

The relative toxicity of Bt rice pollen to domestic silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae), was assessed by a leaf-dip bioassay under laboratory conditions. Silkworm first instars were sensitive to pollen from Bt rice lines, B1 and KMD1, but were not sensitive to pollen from Bt rice line TT9-3. First instars were 1.34-2.12 times more sensitive to B1 pollen than older instars. Bioassays of subacute toxicity under a worst-case scenario suggested that continuous exposure to a sublethal dose of B1 pollen or equivalent doses of non-Bt rice pollen affected silkworm survival and development. Young larvae were more affected by continuous exposure to Bt pollen than older larvae but less affected by non-Bt pollen. Ultrastructural observations showed that Cry proteins associated with Bt pollen were released into the larval lumen and resulted in pathological midgut changes and negative impacts on silkworm survival and development. However, considering that the sublethal dose of Bt pollen (LC15) used in this study is equivalent to the highest detected density of rice pollen on mulberry leaf under field conditions and that the likelihood of such high density of rice pollen occurring in the fields is extremely low, we suggest that the risk of Bt rice pollen on silkworm rearing is negligible.

Complete sequence and organization of Antheraea pernyi nucleopolyhedrovirus, a dr-rich baculovirus.

BACKGROUND: The completion and reporting of baculovirus genomes is extremely important as it advances our understanding of gene function and evolution. Due to the large number of viral genomes now sequenced it is very important that authors present significantly detailed analyses to advance the understanding of the viral genomes. However, there is no report of the Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) genome. RESULTS: The genome of AnpeNPV, which infects Chinese tussah silkworm (Antheraea pernyi), was sequenced and analyzed. The genome was 126,629 bp in size. The G+C content of the genome, 53.4%, was higher than that of most of the sequenced baculoviruses. 147 open reading frames (ORFs) that putatively encode proteins of 50 or more amino acid residues with minimal overlap were determined. Of the 147 ORFs, 143 appeared to be homologous to other baculovirus genes, and 4 were unique to AnpeNPV. Furthermore, there are still 29 and 33 conserved genes present in all baculoviruses and all lepidopteran baculoviruses respectively. In addition, the total number of genes common to all lepidopteran NPVs is sill 74, however the 74 genes are somewhat different from the 74 genes identified before because of some new sequenced NPVs. Only 6 genes were found exclusively in all lepidopteran NPVs and 12 genes were found exclusively in all Group I NPVs. AnpeNPV encodes v-trex(Anpe115, a 3' to 5' repair exonuclease), which was observed only in CfMNPV and CfDEFNPV in Group I NPVs. This gene potentially originated by horizontal gene transfer from an ancestral host. In addition, AnpeNPV encodes two conotoxin-like gene homologues (ctls), ctl1 and ctl2, which were observed only in HycuNPV, OpMNPV and LdMNPV. Unlike other baculoviruses, only 3 typical homologous regions (hrs) were identified containing 2~9 repeats of a 30 bp-long palindromic core. However, 24 perfect or imperfect direct repeats (drs) with a high degree of AT content were found within the intergenic spacer regions that may function as non-hr, ori-like regions found in GrleGV, CpGV and AdorGV. 9 drs were also found in intragenic spacer regions of AnpeNPV. CONCLUSION: AnpeNPV belongs to Group I NPVs and is most similar to HycuNPV, EppoNPV, OpMNPV and CfMNPV based on gene content, genome arrangement, and amino acid identity. In addition, analysis of genes that flank hrs supported the argument that these regions are involved in the transfer of sequences between the virus and host.

Expression of open reading frames in silkworm pupal cDNA library.

A cDNA library containing 2409 singletons was constructed from whole silkworm pupae (Bombyx mori) In addition, the types of genes overexpressed in pupa were analyzed. These genes contained 79 types of proteins with the exception of enzyme, mitochondrial DNA, andribosomal protein. Also analyzed were the expression and nonexpression of open reading frame (ORF) sequences in Escherichia coli. cDNA sequences were compared to the silkworm (B. mori) genome in the GenBank database and the silkworm cDNA database including the SilkBase and KAIKOBLAST databases and 498 novel expressed sequence tags (ESTs) and 217 unknown ESTs were found. After comparison with all available ORF-complete mRNA sequences from the same organism (fruitfly, mosquito, and apis) in the RefSeq collection, 1659 full-length cDNA were identified. In addition, the structure of silkworm mRNA was analyzed, and it was found that 66.8% of silkworm mRNA tailed with poly(A) contained the highly conserved AAUAAA signal and the signal located 10-17 nucleotides upstream of the putative poly(A). Finally, the composition of nucleotides in promoter region for all ESTs was surveyed. The results imply that the TTTTA box may possess some functions in regulating transcription and expression of some genes.

Density functional studies on the endohedral complex of fullerene C70 with tetrahedrane (C4H4): C4H4@C70.

B3LYP/6-31G(d) hybrid HF/DFT calculations were carried out to determine the structural and electronic properties of the endohedral complex of a C(70) cage with tetrahedrane (C(4)H(4)). It was demonstrated that the formation of the complex is endothermic, with a destabilization energy of 72.56 kcal mol(-1). C(4)H(4) is seated in the center of the C(70) cage and exists in molecular form inside the fullerene. C(4)H(4) endohedral doping slightly perturbs the molecular orbitals of C(70). The calculated HOMO-LUMO gaps, the electron affinity (EA), and the ionization potential (IP) indicate that C(4)H(4)@C(70) is more chemically reactive than C(70). The IR active modes and harmonic vibrational frequencies of C(4)H(4)@ C(70) are also discussed.

Using chimeric piggyBac transposase to achieve directed interplasmid transposition in silkworm Bombyx mori and fruit fly Drosophila cells.

The piggyBac transposon has been long used to integrate foreign DNA into insect genomes. However, undesirable transgene expression can result from random insertions into the genome. In this study, the efficiency of chimeric Gal4-piggyBac transposase in directing integration onto a DNA target plasmid was evaluated in cultured silkworm Bombyx mori Bm-12 and fruit fly Drosophila Schneider 2 (S2) cells. The Gal4-piggyBac transposase has a Gal4 DNA-binding domain (DBD), and the target plasmid has upstream activating sequences (UAS) to which the Gal4 DBD can bind with high affinity. The results indicate that, in the Bm-12 and S2 cells, transpositional activity of Gal4-piggyBac transposase was increased by 4.0 and 7.5 times, respectively, compared to controls, where Gal4-UAS interaction was absent. Moreover, the Gal4-piggyBac transposase had the ability of directing piggyBac element integration to certain sites of the target plasmid, although the target-directing specificity was not as high as expected. The chimeric piggyBac transposase has the potential for use in site-directed transgenesis and gene function research in B. mori.

Endohedral complex of fullerene C60 with tetrahedrane, C4H4@C60.

B3LYP/6-31G(d) hybrid HF/DFT calculations were carried out to determine the structural and electronic properties of the endohedral complex of C60 with tetrahedrane C4H4. It was demonstrated that C4H4 was seated in the center of the C60 cage and existed in a molecular form inside the fullerene. The formation of this complex was endothermic with inclusion energy of 141.05 kcal/mol. C4H4 endohedral doping slightly perturbed the molecular orbitals of C60. The calculated HOMO-LUMO gaps, the electron affinity(EA) and the ionizational potential (IP) indicated that C4H4@C60 seemed to be more kinetically reactive than C60. The IR active modes and harmonic vibrational frequencies of C4H4@C60 were also discussed.