ABSTRACT
BACKGROUND: The oriental fruit fly, Bactrocera dorsalis (Hendel), a very destructive insect pest in many areas of Asia, including Taiwan, can cause significant damage by ovipositing in and larval feeding on many kinds of fruit. A female lethal system, combining the splicing property of doublesex (dsx) with the toxicity of ricin A chain (RTA), has been developed. In this system, a modified RTA is separated by Bddsx intron 3; the expressed RNA can only be spliced in females, with toxic effects, whereas the immature RTA in males is harmless. RESULTS: Two RTA-Bddsx constructs, clone BE 24-7 and clone CF 26-21, containing Bddsx intron 3 and its flanking exonic sequences, with four nucleotides at the 5'-end and five nucleotides at the 3'-end, correctly spliced in a sex-specific manner. Wild-type and modified RTAs expressed in an Escherichia coli system retained their ability to suppress protein synthesis: 90.4% for Ricin-WT, 71.3% for Ricin-LERQ and 58.0% for Ricin-FEGQ. Embryonic injection of Acp-CF26-21, the RTA-Bddsx gene driven by the actin 5C promoter, resulted in a significant increase in male percentage in the eclosed adults. CONCLUSION: Our results indicate that the RTA-Bddsx hybrid system offers a novel and promising approach for oriental fruit fly control.
Subject(s)
Insect Proteins/genetics , Plant Diseases/parasitology , Plants/parasitology , Ricin/genetics , Tephritidae/genetics , Alternative Splicing , Animals , Female , Fruit/parasitology , Insect Proteins/metabolism , Larva , Male , Oviposition , Promoter Regions, Genetic/genetics , Recombinant Fusion Proteins , Ricin/metabolism , Sex Ratio , Tephritidae/physiologyABSTRACT
Ehrlichia (E.) canis is a Gram-negative obligate intracellular bacterium responsible for canine monocytic ehrlichiosis. Currently, the genetic diversity of E. canis strains worldwide is poorly defined. In the present study, sequence analysis of the nearly full-length 16S rDNA (1,620 bp) and the complete coding region (4,269 bp) of the gp200 gene, which encodes the largest major immunoreactive protein in E. canis, from 17 Taiwanese samples was conducted. The resultant 16S rDNA sequences were found to be identical to each other and have very high homology (99.4~100%) with previously reported E. canis sequences. Additionally, phylogenetic analysis of gp200 demonstrated that the E. canis Taiwanese genotype was genetically distinct from other reported isolates obtained from the United States, Brazil, and Israel, and that it formed a separate clade. Remarkable variations unique to the Taiwanese genotype were found throughout the deduced amino acid sequence of gp200, including 15 substitutions occurring in two of five known species-specific epitopes. The gp200 amino acid sequences of the Taiwanese genotype bore 94.4~94.6 identities with those of the isolates from the United States and Brazil, and 93.7% homology with that of the Israeli isolate. Taken together, these results suggest that the Taiwanese genotype represents a novel strain of E. canis that has not yet been characterized.
Subject(s)
Bacterial Proteins/genetics , Ehrlichia canis/classification , Ehrlichia canis/genetics , Phylogeny , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Dogs , Genotype , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , Sequence Alignment , Sequence Analysis, Protein , TaiwanABSTRACT
The genetic diversity of Babesia gibsoni strains worldwide is currently poorly defined. The aim of the present study was to characterize B. gibsoni strains in naturally infected dogs in Taiwan using a combination of polymerase chain reaction (PCR) and sequence analysis of both 18S rDNA and the gene encoding thrombospondin-related adhesive protein (TRAP). Genomic DNA was extracted from 29 parasitemic dogs, and the target genes were separately amplified, sequenced and aligned with corresponding sequences available in GenBank. All 18S rDNA sequences (1,262 bp) amplified from the Taiwanese isolates were identical to each other and had very high similarity (99.9-100%) with previously reported B. gibsoni sequences. These results provide the first molecular evidence showing infection of dogs with B. gibsoni from Taiwan. On the other hand, a phylogenetic analysis based on the deduced amino acid sequence of the TRAP gene demonstrated that the Taiwanese isolates were closely related to strains previously identified from Okinawa Island, Japan, but genetically distinct from strains found on Honshu in Japan and Jeju Island in South Korea. The divergence of TRAP among the geographically dispersed strains examined in this study and others supports the conclusion that this gene is useful for molecular genotyping of B. gibsoni strains.
Subject(s)
Babesia/genetics , Babesiosis/veterinary , Dog Diseases/parasitology , Protozoan Proteins/genetics , Amino Acid Sequence , Animals , Babesia/isolation & purification , Babesiosis/blood , Babesiosis/genetics , Base Sequence , DNA Primers/genetics , Dogs , Gene Amplification , Molecular Sequence Data , Polymerase Chain Reaction , Protozoan Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , TaiwanABSTRACT
The event-specific real-time detection and quantification of Roundup Ready soybean (RRS) using an ABI PRISM 7700 sequence detection system with light upon extension (LUX) primer was developed in this study. The event-specific primers were designed, targeting the junction of the RRS 5' integration site and the endogenous gene lectin1. Then, a standard reference plasmid was constructed that carried both of the targeted sequences for quantitative analysis. The detection limit of the LUX real-time PCR system was 0.05 ng of 100% RRS genomic DNA, which was equal to 20.5 copies. The range of quantification was from 0.1 to 100%. The sensitivity and range of quantification successfully met the requirement of the labeling rules in the European Union and Taiwan.
