Abstract: 【Aim】The aim of this study is to identify the upstream regulatory sequence of the male determining factor gene MoY of the melon fly, Zeugodacus cucurbitae， and to explore the impact of this sequence on the sex determination of Z. cucurbitae by directing the expression of MoY in the early embryonic stages, so as to provide a reference basis and available elements for the construction of the subsequent Z. cucurbitae transgenic strains.【Methods】We amplified and sequenced the upstream sequence of MoY linked to the Y chromosome of Z. cucurbitae using the genome-walking, and connected the obtained upstream sequence to the CDS region of MoY to construct a plasmid that drives the expression of MoY. We injected the plasmid expressing MoY into the fresh embryos of Z. cucurbitae, and after hatching, extracted the genomic DNA based on the observed adult phenotype and sex ratio to amplify MoY so as to determine whether the upstream regulatory sequence of MoY had the activity of directing MoY expression and analyze the impact of MoY expression in embryos on the gender determination. 【Results】The upstream sequence of 1 660 bp of MoY of Z. cucurbitae was cloned and obtained, and the plasmid p1660 that drives the MoY expression was constructed. Eighteen male and 13 female adults developed from the injected embryos of Z. cucurbitae, and three adults were found to have abnormal genitalia, with negative amplification results for MoY, confirming them as intersex individuals. 【Conclusion】In this study, we found that the upstream regulatory sequence of MoY of Z. cucurbitae obtained by genome-walking amplification has the activity of directing the expression of MoY. When MoY is expressed in the early stages of Z. cucurbitae embryos, the gender reversal in the previously developed female individuals can be happened.

Key words: Zeugodacus cucurbitae, MoY, upstream regulatory sequence, gender reversal, intersex individual