Isolation and Molecular Characterization of the Transformer Gene From Bactrocera cucurbitae (Diptera: Tephritidae)

Abstract

transformer (tra) is a switch gene of sex determination in many insects, particularly in Dipterans. However, the sex determination pathway in Bactrocera cucurbitae (Coquillett), a very destructive pest on earth, remains largely uncharacterized. In this study, we have isolated and characterized one female-specific and two male-specific transcripts of the tra gene (Bcutra) of B. cucurbitae. The genomic structure of Bcutra has been determined and the presence of multiple conserved Transformer (TRA)/TRA-2 binding sites in Bcutra has been found. BcuTRA is highly conservative with its homologues in other tephritid fruit flies. Gene expression analysis of Bcutra at different developmental stages demonstrates that the female transcript of Bcutra appears earlier than the male counterparts, indicating that the maternal TRA is inherited in eggs and might play a role in the regulation of TRA expression. The conservation of protein sequence and sex-specific splicing of Bcutra and its expression patterns during development suggest that Bcutra is probably the master gene of sex determination of B. cucurbitae. Isolation of Bcutra will facilitate the development of a genetic sexing strain for its biological control.

Keywords: Bactrocera cucurbitae (Coquillett); Bcutra; sex determination; sex-specific splicing; transformer.

Fig. 1.

(A) Genomic structure and transcripts of Bcutra. (B) The consensus sequences of the putative TRA/TRA-2 binding sites, TRA-2 ISS and RBP1 binding sites. (A) Exons and introns of Bcutra are represented by boxes and lines, respectively. Exon and intron sizes are represented by the numbers in the boxes and under the lines, respectively. Exons 1A, 1B, 2, 3, and 4 (blue) are common in both females and males. Exons MS1, 2, and 3 are male specific. The start codon and the first stop codon in exons are labeled by black vertical bars. The positions of the putative TRA/TRA-2 binding sites, TRA-2 ISS, and RBP1 binding sites are labeled by purple, yellow, and green vertical bars, respectively. The diagram is not drawn to scale. (B) The conserved sequences are bolded.

Fig. 2.

Multiple sequence alignment and phylogenetic tree of TRA proteins. (A) Multiple sequence alignment of TRA proteins in B. cucurbitae, B. dorsalis, B. oleae, B. jarvisi, B. tryoni, B. correcta, A. suspensa, C. capitata, and D. melanogaster. The black-shaded represent the amino acids that are identical in all species, and the gray-shaded represent that the amino acid similarity between species is higher than 75%. The boundary of exons is shown by red vertical lines. SR are within the red box. (B) The neighbor-joining distance tree of Dipteran TRA proteins. The numbers next to the branches represent bootstrap support values from 1,000 replicates. The scale represents the mean character distance. The families are exhibited by color bars on the right. B. cucurbitae (this study) is highlighted with red and with the red triangle. A. obliqua (GenBank: ABW04165.1), A. ludens (GenBank; ABW04176.1), B. dorsalis (GenBank: AME17913.1), B. oleae (GenBank: CAG29241.1), B. tryoni (GenBank: AIK25402.1), B. jarvisi (GenBank: AIK25400.1), C. capitata (GenBank: AAM88673.1), C. macellaria (GenBank: AGE31794.1), C. hominivorax (GenBank: AGE31793.1), D. melanogaster (GenBank: AAB59226.1), D. sechellia (GenBank: AAO38911.1), L. sericata (GenBank: AGE31795.1), L. cuprina (GenBank: ACS34687.2), M. domestica (GenBank: ACY40709.1).

Fig. 3.

Expression of Bcutra at different developmental stages. (A) The exon and intron structure of Bcutra. The primers used in (B) and (C) are labeled. (B) The male and female transcripts are amplified using RT-PCR with the primers F11+ and R1573-. (C) The male-specific transcripts are amplified using RT-PCR with the primers F311+ and R1063-. M: DL1000 DNA marker; E0.5-E8: embryos of mixed sexes at 0–0.5, 0.5–1, 1–3, 3–6, 6–12, 12–24, and 24–48 h after egg laying; L1–L3: the first, second and third instar larvae of mixed sexes; P: pupae of mixed sexes; ♂: male adults; ♀: female adults. α-tub: the internal control gene α-tubulin.

Fig. 4.

Model for sex determination in B. cucurbitae. The functional proteins are represented with color, and the nonfunctional proteins are colorless. Female-specific, male-specific, and common proteins are labeled with blue, green, and orange, respectively.