斜纹夜蛾种群的遗传调控
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斜纹夜蛾(Spodoptera litura)从属于鳞翅目，夜蛾科，是一种重要的世界性广泛分布的农业害虫，国外地区主要分布在非洲、亚热带区域、地中海区域、东南亚及南亚区域，国内主要分布在长江流域和黄河流域。目前针对斜纹夜蛾的防治还是以化学农药为主，但随着害虫抗药性以及农药安全性问题的加剧，斜纹夜蛾新型防治方法的需求日渐明显，昆虫种群的遗传调控技术是斜纹夜蛾害虫防治的理想选择。本研究基于昆虫种群遗传调控技术的原理，试图从昆虫转基因技术和基因组编辑技术着手，期望构建斜纹夜蛾种群遗传调控体系。昆虫转基因技术是昆虫遗传调控技术的基础。在本实验中，我们构建了基于piggyBac转座子的转基因质粒。经过对斜纹夜蛾产卵特性的深入探究，我们建立并优化了斜纹夜蛾胚胎显微注射平台。在本研究中，我们注射了Oxitec4319， IE1-DsRed，3XP3-EGFP和IE1-DsRed-ovoP-1.2k-EGFP 4种转基因质粒，在胚胎显微注射48h后，能够观察到报告基因很强的瞬时表达效果，这说明这些报告基因可以在斜纹夜蛾胚胎中表达相应的蛋白并发生作用。以上的工作为斜纹夜蛾转基因研究奠定了理论基础和实践依据。基因组编辑技术是昆虫遗传调控的新方向，更是基因功能研究的新方法。CRISPR/Cas9技术为昆虫基因组功能研究提供了有效的工具。最近由CRISPR/ Cas9系统介导的靶基因的突变已经在双翅目，鳞翅目和鞘翅目等昆虫纲中的几个目中得以实现。然而，由于缺乏基因组信息和胚胎显微注射技术，这一系统在农业害虫中的应用却鲜有报道。在本研究中，我们证明了CRISPR/Cas9系统能够有效的引起鳞翅目害虫斜纹夜蛾相应基因的突变。我们成功的突变了斜纹夜蛾重要的胚胎发育的基因abd-A (Slabd-A)基因，并确定它在昆虫腹节的形成方面发挥了显著作用。直接注射Cas9 mRNA和Slabd-A特异的sgRNA至斜纹夜蛾胚胎中能够成功获得典型的abd-A缺陷表型，即在幼虫期体节的异常和色素沉着的异位。基于PCR的分析表明，Cas9/sgRNA复合体能够有效地诱导斜纹夜蛾靶标位点的突变。综上所述，CRISPR/Cas9系统是在鳞翅目害虫斜纹夜蛾的基因组操作方面是一个有力的工具，并为斜纹夜蛾的遗传防治提供了新思路。
The tobacco cutworm, Spodoptera litura, belongs to the order Lepidoptera, family Noctuidae, and is an important globally widespread agricultural pest. It is mainly distributed abroad in Africa, subtropical regions, the Mediterranean region, Southeast Asia and South Asia, and in China, it is primarily found in the Yangtze River and Yellow River basins. Currently, chemical pesticides remain the main method for controlling S. litura, but with the worsening of pest resistance and pesticide safety issues, the demand for novel control methods for this pest has become increasingly prominent. Insect population genetic control technology is an ideal option for the management of S. litura. Based on the principles of insect population genetic control technology, this study attempts to start from insect transgenic technology and genome editing technology, with the aim of constructing a population genetic control system for S. litura. Insect transgenic technology is the foundation of insect genetic control technology. In this experiment, we constructed transgenic plasmids based on the piggyBac transposon. After an in-depth investigation of the oviposition characteristics of S. litura, we established and optimized an embryonic microinjection platform for this pest. In this study, four transgenic plasmids, namely Oxitec4319, IE1-DsRed, 3XP3-EGFP, and IE1-DsRed-ovoP-1.2k-EGFP, were injected. At 48 hours after embryonic microinjection, strong transient expression of the reporter genes could be observed, indicating that these reporter genes can express corresponding proteins and function in the embryos of S. litura. The above work lays a theoretical and practical foundation for transgenic research on S. litura. Genome editing technology is a new direction for insect genetic control, as well as a novel method for gene function research. The CRISPR/Cas9 system provides an effective tool for the study of insect genome function. Recently, target gene mutations mediated by the CRISPR/Cas9 system have been achieved in several orders of Insecta, including Diptera, Lepidoptera and Coleoptera. However, due to the lack of genomic information and embryonic microinjection technology, the application of this system in agricultural pests has rarely been reported. In this study, we demonstrated that the CRISPR/Cas9 system can effectively induce mutations of corresponding genes in the lepidopteran pest S. litura. We successfully mutated the important embryonic development gene abd-A (Slabd-A) of S. litura, and confirmed that it plays a significant role in the formation of insect abdominal segments. Direct injection of Cas9 mRNA and Slabd-A-specific sgRNA into S. litura embryos successfully obtained typical abd-A defective phenotypes, namely abnormal body segments and ectopic pigmentation in the larval stage. PCR-based analysis showed that the Cas9/sgRNA complex can effectively induce mutations at the target sites of S. litura. In summary, the CRISPR/Cas9 system is a powerful tool for genomic manipulation of the lepidopteran pest S. litura, and provides new ideas for the genetic control of this pest.
