ABSTRACT
dsRNA is a powerful tool for gene-specific silencing in plants and animals. In this study, we examined the use of gene silencing in generating transgenic silkworms resistant to the Bombyx mori nucleopolyhedrovirus (BmNPV). Using a transposon piggyBac system, we first generated BmN cells (rBmN-lef1), which carried artificial genes designed for expressing dsRNAs with sequences of the essential viral gene lef-1. NPV DNA microarray analysis revealed that the accumulation of lef-1 mRNA was successfully inhibited in rBmN-lef1 infected with BmNPV. The virus titer in the culture medium of rBmN-lef1 at 48 hr post-infection (h.p.i.) was 50% of that of the control cells. Moderate BmNPV-resistance caused by transgenesis of the artificial dsRNA-expressing gene was confirmed in the transgenic silkworms. Virus production was reduced in transgenic silkworms relative to controls up to 96 hrs after viral inoculation. Although complete protection was not achieved and the transgenic larvae ultimately died, this is the first report to show the use of RNAi in confering enhanced viral resistance on transgenic animals.
Subject(s)
Bombyx/virology , Nucleopolyhedroviruses/physiology , RNA Interference , Animals , Animals, Genetically Modified , Bombyx/genetics , Gene Expression Profiling , Genes, Essential , Genes, Viral , Oligonucleotide Array Sequence Analysis , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Messenger/analysis , RNA, Viral/analysis , Viral Plaque Assay , Viral Proteins/genetics , Virus ReplicationABSTRACT
Bombyx mori densonucleosis virus type 2 (BmDNV-2) is a small, spherical virus containing two complementary single-stranded linear DNA molecules (VD1, VD2). BmDNV-2 is a new type of virus with a unique, yet unspecified replication mechanism which is different from that of parvoviruses (Bando, H., Choi, H., Ito, Y., Nakagaki, M. , Kawase, S., 1992. Structural analysis on the single-stranded genomic DNAs of the virus newly isolated from silkworm: the DNA molecules share a common terminal sequence, Arch. Virol. 124, 187-193; Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M. , Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Hayakawa, T., Asano, S., Sahara, K., Iizuka, T., Bando, H., 1997. Detection of replicative intermediate with closed terminus of Bombyx densonucleosis virus. Arch. Virol. 142, 1-7). Recent analyses on the genomic information of BmDNV-2 identified open reading frames which code for three tentative nonstructural proteins and four (VP1 to 4) of the six known structural proteins (Bando, H., Hayakawa, T., Asano, S., Sahara, K., Nakagaki, M., Iizuka, T., 1995. Analysis of the genetic information of a DNA segment of a new virus from silkworm, Arch. Virol., 140, 1147-1155; Nakagaki et al., in preparation). In this report we demonstrate that the two largest ORFs, VD1-ORF1 and VD2-ORF1, code for the two remaining structural proteins. In addition, computer-assisted analysis revealed that the structural protein encoded in VD1-ORF1 contains sequences conserved among various DNA polymerases, and showed an evolutionary relationship with the DNA polymerases involved in protein-primed replication.
Subject(s)
Bombyx/virology , DNA-Directed DNA Polymerase/genetics , Densovirus/chemistry , Densovirus/genetics , Viral Structural Proteins/genetics , Amino Acid Motifs , Amino Acid Sequence , Animals , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Densovirus/isolation & purification , Densovirus/metabolism , Genome, Viral , Molecular Sequence Data , Phylogeny , Sequence Analysis, DNA , Viral Structural Proteins/chemistry , Viral Structural Proteins/metabolismABSTRACT
A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.
