The Isolation And Molecular Characterization Of The Transformer Gene From Melon Fly Bactrocera Cucurbitae (Dipteran: Tephritidae)

Bactrocera cucurbitae (Copuillett) is a quarantine pest that is classified into Bactrocera Macquar, family Tephritidae, order Diptera., With strong fecundity and wide parasitic range, they mainly harm Cucurbitaceae and Solanaceae plants. It has become one of the major pests of agricultural production worldwide. Sex determination is a key factor affecting their reproduction and developments. transformer(tra) is a key switch gene in sex determination of many insts, particularly in Tephritidae family. However, the molecular mechanism of sex determination in B. cucurbitae remains unclear and the transformer gene in B. cucurbitae has not been isotated yet.In this study, the Bactrocera cucurbitae as experiment materials, Using RT-PCR combined with RACE PCR strategy was performed to isolate Bctra gene , To adopt semi-quantitative RT-PCR methods to study the expression of tra gene in different developmental stage of B. cucurbitae, and discusses the possible role of sex determination genes in ctrocera cucurbitae,Then,CRISPR/Cas9 technique was used for knockout for Bcutra , in an effort provide new ideas and new methods for the prevention and control of Bactrocera cucurbitae.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 TRA/TRA-2 binding sites in Bcutra has been found. BcuTRA is highly conserved with TRA proteins 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 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 determinationIn addition, we have synthesized Cas9 mRNA and sgRNA mRNA of Bcutra to knock out the gene to farther its roles in sex determination, our research will facilitate the development of control pests using biological engineering methods.