桔小实蝇雌性特异胚胎致死品系的构建

发布时间：2018-04-15 14:04

本文选题：桔小实蝇 + 雌性特异致死品系　；参考：《中国农业科学院》2015年博士论文

【摘要】：昆虫不育技术(Sterile insect technique,SIT)是根除实蝇类害虫和预防害虫在高风险地区定殖的最有效的防治方法。本研究以世界性农业害虫桔小实蝇Bactrocera dorsalis(Hendel)为对象,通过分离、鉴定桔小实蝇雌性特异胚胎致死的遗传性别品系特异调控元件,构建桔小实蝇雌性特异胚胎致死驱动载体和效应载体,建立桔小实蝇遗传转化技术,以期获得桔小实蝇雌性特异胚胎致死品系,实现在桔小实蝇饲养阶段清除所有雌虫,为不育雄虫释放的昆虫不育技术项目提供技术支撑。主要研究结果如下:(1)利用实时荧光定量PCR分析Bdvasa的时空表达谱,反向PCR分离Bdvasa 5’侧翼的上游启动子序列,结果表明Bdvasa是一种在胚胎早期高水平表达的母体效应基因,分离得到Bdvasa翻译起始密码子上游1996 bp序列,成功用于构建驱动t TA表达的驱动载体2个。(2)为分离候选的桔小实蝇显性致死基因,通过同源基因克隆和RACE技术克隆桔小实蝇细胞凋亡效应酶caspase-1,实时荧光定量PCR分析该基因的时空表达谱,腹部RNAi研究该基因在雌虫生殖过程中的功能,结果为:克隆得到桔小实蝇细胞凋亡效应酶caspase-1的c DNA全长1679 bp,编码328个氨基酸残基,并将该基因命名为Bdcp-1;Bdcp-1在桔小实蝇的不同发育时期和成虫的不同组织间均有表达,其表达量与桔小实蝇生长发育过程密切相关;适量的UV照射可以提高Bdcp-1 m RNA转录水平,Bdcp-1在UV照射1 h的8日龄蛹中的表达量是未照射的5.6倍,照射1.5 h是未照射的5.2倍,暗示细胞通过caspase级联反应调控受损细胞凋亡,维持细胞正常的生命活动,但照射超过2 h后,这种凋亡机制被破坏;Bdcp-1在雌虫生殖过程中具有重要作用,该基因沉默后,卵巢发育和卵黄蛋白基因Bdyp1的表达受到抑制,产卵前期延长,单头雌虫累计产卵量显著降低。(3)为了分离桔小实蝇性别特异剪接调控元件,利用PCR结合RACE技术分析桔小实蝇Bdtra和Bdtra-2的基因组和c DNA结构,半定量PT-PCR研究这2个基因的表达模式,胚胎RNAi研究这2个基因的功能。结果为:桔小实蝇Bdtra存在1个雌性特异剪接体和2个雄性特异剪接体,Bdtra-2不存在性别特异剪接;Bdtra雌性特异剪接体和Bdtra-2基因具有母体遗传表达特性,Bdtra雄性特异剪接体在产后1 h后的胚胎中才能检测到;胚胎RNAi Bdtra或Bdtra-2均能高效诱导雌性胚胎发育为表型雄虫,证明Bdtra和Bdtra-2在雌虫体性发育过程中具有重要作用;分离得到Bdtra雌性特异剪接内含子序列Bdtra I1068 bp,成功用于构建雌性特异致死效应载体。(4)利用胚胎显微注射的方法,遗传转化地中海实蝇精子荧光标记质粒1260,荧光显微镜筛选得到2种不同荧光表达模式的桔小实蝇转化个体,RT-PCR检测到荧光蛋白基因t GEP和Ds Red在转化个体中有表达,成功建立桔小实蝇遗传转化技术体系。
[Abstract]:Sterile insect technique site is the most effective control method for the eradication of fly pests and the prevention of colonization of insect pests in high-risk areas.In this study, the worldwide agricultural pest Bactrocera dorsalis Hendeler was used to identify the genetic and sex-specific regulatory elements of female specific embryo death.The driving vector and effect vector of female specific embryo death were constructed, and the genetic transformation technology of fruit fly was established in order to obtain the female specific embryo lethal strain of fruit fly and to eliminate all female insects in the rearing stage of fruit fly.To provide technical support for the male sterile male release insect sterility technology project.The main results are as follows: (1) using real-time fluorescence quantitative PCR to analyze the temporal and spatial expression profiles of Bdvasa, reverse PCR was used to isolate the upstream promoter sequence of Bdvasa 5'flanking. The results showed that Bdvasa was a high level maternal effector gene expressed at early embryonic stage.The upstream 1996 BP sequence of Bdvasa translation initiation codon was isolated and successfully used to construct two driving vectors for TTA expression.The apoptotic effector enzyme caspase-1 was cloned by homologous gene cloning and RACE technique. The temporal and spatial expression profiles of the gene were analyzed by real-time fluorescence quantitative PCR, and the function of the gene during female reproduction was studied by abdominal RNAi.The results showed that the total length of c DNA of the apoptosis effector enzyme caspase-1 was 1679 BP, encoding 328 amino acid residues, and Bdcp-1 Bdcp-1 was expressed in different stages of development and in different tissues of adults.It was suggested that the apoptosis of damaged cells was regulated by caspase cascade reaction and maintained normal life activity. However, after irradiation for more than 2 hours, this mechanism of apoptosis was destroyed and Bdcp-1 played an important role in the reproductive process of female worms, and the gene was silenced.The ovarian development and the expression of yolk protein gene Bdyp1 were inhibited, the pre-oviposition was prolonged, the accumulative oviposition of single female decreased significantly.The genomes and c DNA structures of Bdtra and Bdtra-2 were analyzed by PCR and RACE, the expression patterns of these two genes were studied by semi-quantitative PT-PCR, and the functions of these two genes were studied by embryonic RNAi.The results showed that there was one female specific splice and two male specific splicing bodies in Bdtra, and there were no sex specific splicing female splicing bodies and Bdtra-2 genes had maternal genetic expression characteristics.Only one hour after delivery could be detected in embryos.RNAi Bdtra or Bdtra-2 could efficiently induce female embryos to develop into phenotypic males, indicating that Bdtra and Bdtra-2 play an important role in the development of females.Bdtra female specific splicing intron sequence Bdtra I1068 BP was isolated and successfully used to construct female specific lethal effect vector.Fluorescence labeling plasmid 1260 was obtained from transgenic spermatozoa of Drosophila morifolia. Two different fluorescent expression patterns were obtained by RT-PCR. The expression of fluorescent protein genes t GEP and Ds Red in transformed individuals was detected by RT-PCR.The technique system of genetic transformation of fruit fly was established successfully.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S433

【参考文献】