DNA sequences differentially represented in males and females of the oriental fruit fly Bactrocera dorsalis.

摘要

The objective of this dissertation is the isolation of DNA sequences that are differentially represented in males and females of the Oriental fruit fly Bactrocera dorsalis by initiating a molecular characterization of Y chromosome sequences. A method known as Representational Difference Analysis (RDA) was utilized to obtain DNA sequences unique to the B. dorsalis male genome. B. dorsalis male genomic DNA served as the "tester" DNA and female genomic DNA as the "driver". Six RDA products were obtained following two rounds of DNA hybridization and difference enrichment via PCR (Polymerase Chain Reaction). One of these products (RDA product 1) was used to isolate a genomic DNA clone (3.1a) from a B. dorsalis male genomic DNA minilibrary that shows similarity to R1 retrotransposable elements. The presence of R1 elements in Tephritid insects has heretofore been undescribed, but they have been described in the genomes of other Diptera.;Oligonucleotide primers designed for the 3.1 a clone produce different amplification patterns in PCRs of genomic DNA from B. dorsalis males vs. females. PCRs using male DNA produce 325 by and 2.6 kb products and only a 2.6 kb product from female DNA. These products are also produced in PCRs of genomic DNA from B. dorsalis embryos and third instar larvae and in other Bactrocera species.;Both PCR products have regions of sequence similarity to R1 elements. The 2.6 kb product contains a putative 1.7 kb open reading frame (ORF) encoding 583 amino acids. The hypothetical ORF product contains three amino acid motifs found in Drosophila R1 reverse transcriptases. Both sequences are repetitively represented in the B. dorsalis male and female genomes. However, the 325 by male product produces male-specific bands when used as a probe for Southern blots of male and female genomic DNA.;The PCR amplification patterns are consistent with what would be expected if the 2.6 kb and 325 by products originated from the B. dorsalis X and Y chromosomes. Thus, the male-specific sequence is potentially useful as a gateway into the B. dorsalis Y chromosome and as a tool for the characterization of other aspects of the B. dorsalis genome.