瓜实蝇雄性决定因子基因MoY功能及其上游调控序列活性分析  

Analysis of the function and upstream regulatory sequence activity of the male determining factor gene MoY in Zeugodacus cucurbitae(Diptera:Tephritidae)

作　　者：符竣凯 温健 曹凤勤 颜日辉 林先武 FU Jun-Kai;WEN Jian;CAO Feng-Qin;YAN Ri-Hui;LIN Xian-Wu(School of Tropical Agriculture and Forestry(Agricultural and Rural College,Rural Revitalization College),Hainan University,Danzhou 570228,China)

机构地区：[1]海南大学热带农林学院(农业农村学院、乡村振兴学院),儋州570228

出　　处：《昆虫学报》2024年第3期339-345,共7页Acta Entomologica Sinica

基　　金：海南省自然科学基金青年基金项目(322QN256);海南省自然科学基金高层次人才项目(323RC408);国家自然科学基金地区基金项目(32260665)。

摘　　要：【目的】本研究旨在鉴定瓜实蝇Zeugodacus cucurbitae雄性决定因子基因MoY上游调控序列,并探索该序列在胚胎早期驱动MoY表达对瓜实蝇性别决定的影响,为后续的瓜实蝇转基因品系的构建提供参考依据以及可用元件。【方法】使用基因组步移法扩增瓜实蝇Y染色体连锁的MoY上游序列并测序,将获得的上游序列连接MoY的CDS区构建其驱动MoY表达的质粒;将MoY表达质粒注射进瓜实蝇新鲜的胚胎中,待胚胎孵化后观察成虫的表型以及性比并提取基因组DNA,通过使用基因组DNA扩增MoY以判断获得的MoY上游调控序列是否具有驱动MoY表达的功能,同时分析MoY在胚胎中的表达对性别决定的影响。【结果】克隆并获得瓜实蝇MoY上游1660 bp序列,构建该序列驱动MoY表达的质粒p1660。由注射胚胎发育而来的瓜实蝇雄成虫18头和雌成虫13头及另外发现3头成虫的生殖器发育异常且MoY的扩增结果为阴性,确认为间性个体。【结论】本研究通过对MoY进行基因组步移扩增发现所获得的瓜实蝇MoY上游调控序列具有驱动MoY表达的活性,当MoY于瓜实蝇胚胎早期表达可使原本发育为雌性的个体出现性别逆转的现象。【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 1660 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.

关 键 词：瓜实蝇 MoY 上游调控序列 性别逆转 间性个体 

分 类 号：Q786[生物学—分子生物学]