摘要
Doublesex是昆虫性别决定级联通路上的关键基因,其在控制昆虫性别分化中起着重要的作用。本研究旨在克隆、鉴定美洲锥虫病重要媒介红带锥蝽(Triatoma rubrofasciata)的性别决定基因doublesex(Trdsx),分析其不同发育时期的表达谱。基于红带锥蝽基因组数据库,比对Trdsx相关片段,分别以雌雄红带锥蝽cDNA为模板,采用PCR方法克隆Trdsx基因,利用生物信息软件对所得序列进行结构域预测、氨基酸序列比对和进化树分析。根据Trdsx特异性表达引物,利用real time RT-PCR方法研究其在红带锥蝽不同发育时期及雌雄个体的表达谱。从红带锥蝽雌、雄成虫中克隆获得一个雌、雄共有的Trdsx cDNA全长开放性读码框序列,命名为TrdsxB。TrdsxB长度为948 nt,编码315个氨基酸。结构域分析发现TrdsxB包含锌离子结合序列、具有保守的锌螯合残基C2H2C4的DM结构域和参与泛素介导的蛋白水解序列的UBA结构域。TrdsxB在卵中高表达,随后逐渐减少,在四期若虫降至最低,之后再次升高。TrdsxB雌、雄锥蝽中存在表达差异。本研究获得了红带锥蝽性别决定基因doublesex(TrdsxB)的全长开放性读码框序列,TrdsxB具有保守的结构域,其在雌、雄锥蝽体内存在表达差异。本研究结果将为红带锥蝽的性别调控机制和生物防制研究提供理论基础。
Doublesex is a key gene in the sex determination pathway of insects,which plays an important role in controlling the sex differentiation of insects.This study aims to clone and identify the sex-determining gene doublesex(Trdsx)of T.rubrofasciata,an important vector of American trypanosomiasis,and analyze the expression profiles at different developmental stages.Based on the genome database of T.rubrofasciata,the Trdsx-related fragments were aligned.The full-length Trdsx gene was cloned by PCR using the cDNA of male and female T.rubrofasciata as templates.The obtained sequences were subjected to domain prediction,amino acid sequence alignment and phylogenetic tree analysis using bioinformatics software.According to Trdsx specific expression primers,real time RTPCR was used to study the expression profile of Trdsx in different developmental stages and male and female individuals.A full-length open reading frame of Trdsx cDNA sequence was cloned from the male and female adults of T.rubrofasciata,which was named TrdsxB.TrdsxB is 948 nt in length and encodes 315 amino acids.Domain analysis revealed that TrdsxB contained a zinc ion binding sequence,a DM domain with a conserved zinc chelating residue C2H2C4,and a UBA domain involved in ubiquitin-mediated proteolysis.TrdsxB was highly expressed in eggs,and then gradually decreased to the lowest in the fourth stage nymphs,and then increased again.The expression of TrdsxB was significantly different between male and female.In this study,the full-length sequence of TrdsxB shared by the sexdetermining gene doublesex in male and female individuals was obtained.TrdsxB has conserved domains and differential expression in male and female individuals.The results of this study provide a theoretical basis for the study of sex regulation mechanism and biological control of T.rubrofasciata.
作者
田芸嘉
郭云海
张仪
朱丹
李元元
刘琴
TIAN Yunjia;GUO Yunhai;ZHANG Yi;ZHU Dan;LI Yuanyuan;LIU Qin(National Institute of Parasitic Diseases,Chinese Center for Disease Control and Prevention(Chinese Center for Tropical Diseases Research)NHC Key Laboratory of Parasite and Vector Biology National Center for International Research on Tropical Diseases WHO Collaborating Centre for Tropical Diseases,Shanghai 200025,China)
出处
《中国动物传染病学报》
CAS
北大核心
2023年第3期150-157,共8页
Chinese Journal of Animal Infectious Diseases
基金
国家重点研发计划项目(2021YFC2300800,2021YFC2300803)
上海市卫健委临床研究专项(202140215)。
