小菜蛾性别决定基因鉴定与功能研究

Sex determination is a cascade control process. Loss-of-function of sex determination gene could lead to abnormal development or even sterility which shows great potential for pest management. Several sex determination genes have been found in lepidopteran insects, however, their specific functions are still unclear. So far, there is no report about the sex determination gene in the most destructive lepidopteran insect, the diamondback moth (DBM), Plutella xylostella. In this study, the sex-determining genes in DBM will be cloned, and their spatial and temporal expression profiles will be analyzed. The genome-editing technique, CRISPR/Cas9, will be employed to produce the sex determination gene knockout individuals. The phenotypic changes of knockout individuals will be documented via histological and scanning electron microscope observation. Further, the transcriptomes of wild-type and knockout individuals will be compared to find out the differentially expressed genes and validated by qRT-PCR. Successive rearing of the obtained gene knockout strains will be conducted to analysis their genetic stability. This work will contribute to the understanding of the diverse functions of lepidopteran insect sex determination gene, as well as provide the important reference basis for the application of CRISPR/Cas technique in the genetic control of pests.

性别决定是一个阶梯式的调控过程。性别决定基因的缺失会造成个体发育异常甚至不育，具有应用于害虫防治的潜力。鳞翅目昆虫中已发现了多种性别决定相关的基因，但具体功能不是十分清楚。目前尚无关于鳞翅目害虫小菜蛾性别决定基因的报道。本研究拟通过克隆小菜蛾性别决定基因，分析其时空表达模式；利用CRISPR/Cas9基因组编辑技术对此类基因进行敲除获得敲除突变体，通过组织学观察和扫描电镜观察分析敲除突变体的表型变化；通过比较分析突变体和野生型的RNA-Seq测序结果，筛查并验证受性别决定基因调控的潜在靶标基因，初步分析小菜蛾性别决定基因的功能；传代饲养敲除突变品系，分析遗传稳定性。本研究将有助于深入解析鳞翅目昆虫性别决定基因的具体功能，并为CRISPR/Cas9技术应用于害虫的遗传防治提供重要的参考依据。

小菜蛾（Plutella xylostella）是世界范围内危害十字花科作物的重要害虫。昆虫性别决定基因具有应用于害虫防治的潜力。目前尚无小菜蛾性别决定基因研究的报道。我们克隆了小菜蛾性别决定基因：P-element somatic inhibitor（Psi）、IGF-II mRNA-binding protein（Imp）、doublesex （Dsx）、transformer2（Tra2）、intersex（Ix）、Sex lethal（Sxl）和Masculization gene（Masc）。通过检测胚胎早期Dsx和Masc可变剪切体的表达情况，确定小菜蛾胚胎期性别决定完成时间在4-5小时。我们利用CRISPR/Cas9技术获得小菜蛾PxPsi和PxDsx敲除突变体。雄性PxPsi敲除突变体中可检测到雌性PxDsx可变剪切体表达，并且外生殖器和精巢发育异常。雌性Psi敲除突变体表型正常。雄性PxPsi敲除突变体中雌性特异Vg基因显著表达，但雄性偏好表达的PBP1和OR1显著下调表达。通过sgRNA靶向雌性特异PxDsx外显子、雄性特异Dsx外显子和雌雄共有2号外显子区域，获得了三种PxDsx敲除突变体。敲除PxDsx基因共同2号外显子，雌、雄突变个体均出现突变表型；雌性特异Vg基因在雄性突变体中显著表达，在雌性突变体中表达量显著下调，而雄性偏好表达的PBP1、OR1和OR3基因在雄性突变体中显著下调，在雌性突变体中表达量显著上调。敲除PxDsx雌性剪接体，雄性突变体正常，雌性突变体出现突变表型；雌性突变体中Vg基因表达量显著下调，而PBP1、OR1和OR3基因表达量显著上调。敲除PxDsx雄性剪接体，雌性突变体正常，雄性突变体出现突变表型；雄性突变体中Vg基因表达量显著上调，而PBP1、OR1和OR3基因表达量显著下调。杂交实验显示，PxDsx共同区域敲除突变会导致雌、雄虫均不育；PxDsx雌性剪接体敲除突变会导致雌虫不育，但雄虫无影响；PxDsx雄性剪接体敲除突变会导致雄虫不育，但雌虫无影响。我们尝试在小菜蛾中建立双系转基因CRISPR/Cas9体系。我们首先获得了sgRNA转基因小菜蛾品系，正在尝试获得Cas9转基因品系。本研究阐明了部分小菜蛾性别决定基因的功能，为利用基因组编辑技术和转基因技术建立小菜蛾的遗传调控体系提供坚实的基础。