光敏感madd-4基因对松材线虫胚胎致死的调控机理研究

Pine wilt disease (PWD) is a quarantine disease damaging several pine tree species in worldwide, with particular respect in Asian countries, like China and Japan. In the past several decades, numerous studies have been done in taxonomy, genetics and behavioral ecology of pinewood nematode, Bursaphelenchus xylophilus. However, little is known about its reproduction and development at molecular level. Our.previous study showed that blue light inhibited gene expression of madd-4, leading to lethality of B. xylophilus during embryogenesis. The embryogenesis of B. xylophilus occurs inside the trunk of pine tree in nature condition, where there is no negative influence from light. However, studying on the lethality mechanism of blue light against the embryogenesis of B. xylophilus could provide a window for developing novel control measures. This project will demonstrate the gene structure and evolution via cloning and characterization of light sensitive gene of madd-4, profile the gene expression pattern in embryo by mRNA in situ hybridization, quantitative real time PCR, and reporter gene fusions, uncover the function of madd-4 during embryogenesis by RNAi and CRSPR/CAS9, as well as demonstrate gene expression and regulation of madd-4 by promoter analysis in B. xylophilus. The findings of the project will demonstrate the mechanism underlying the madd-4 lethality with blue light in embryo, and explain the reasons why blue light inhibits the population growth and propagations of B. xylophilus. Our findings will also provide the theoretical principles to design specific agents against pine wilt disease at molecular level, and could be used as reference for related research on other organisms.

迄今为止，国内外在松材线虫生殖发育方面的研究比较少见。前期研究发现，光照可以抑制松材线虫madd-4基因的表达，并导致胚胎滞育和死亡。在自然条件下，松材线虫的胚胎发育在树体内完成，不会受到光照的影响，但通过对光敏感madd-4基因调控胚胎致死的作用机理研究，能够从生殖发育角度为探索抑制松材线虫种群增长的控制措施提供理论基础。本项目拟在前期研究的基础上，分析松材线虫光敏感madd-4基因的序列，明确其进化保守性；通过原位杂交、实时定量PCR和转基因研究，阐明其在松材线虫胚胎期的表达模式；通过RNAi和CRSPR/CAS9验证其功能作用，并从启动子角度分析madd-4基因的表达调控，揭示光照抑制松材线虫胚胎发育的内在机制。研究结果不仅能够揭示madd-4基因在松材线虫胚胎致死中的调控机理，为研制新型杀线剂和设计特异性的防控措施提供新的思路，也能够为其它高等动物的胚胎发育和相关疾病研究提供参考。

松材线虫病是一种由松材线虫引起的检疫性病害。前期研究发现，蓝光可以显著地抑制松材线虫的胚胎发育；进一步研究表明，在蓝色光照条件下，很多与代谢有关的基因表达量显著下调，其中madd-4基因的下调幅度尤为显著。在上述研究的基础上，本项目以madd-4基因为研究对象，克隆了madd-4基因，分析该基因的进化地位；构建了madd-4基因相应的质粒表达载体，通过组织原位杂交和转基因显微注射研究，阐明其在松材线虫整个生命周期中的表达模式；通过RNAi技术验证其在胚胎期的功能作用。研究结果不仅能够阐明madd-4基因在松材线虫胚胎发育过程中的调控作用，为揭示光照抑制松材线虫种群增长的机理提供基础，还能够为研制新型杀线剂和设计特异性的松材线虫病防控措施提供新的思路，同时也能为其它高等动物的胚胎发育研究提供一定的参考。本项目主要研究结果如下：.一、madd-4基因在结构上与秀丽隐杆线虫相似，均有madd-4-long和madd-4-short两种不同形式的转录本，两种线虫的两个转录本均共享madd-4-short的第2-10个外显子，说明该基因在两种线虫中可能具有相同的调控功能。.二、in situ原位杂交和转基因显微注射研究结果表明，madd-4基因在松材线虫的胚胎、二龄、三龄、四龄和成虫等整个生命周期内专一地在咽部中枢神经环及背腹神经索中的运动神经元细胞中稳定表达；.三、RNAi浸泡实验结果表明，松材线虫madd-4基因在胚胎期具有三个方面的功能，一是负责调控虫体肌肉臂向中枢神经环和背腹神经索的延伸进程，二是负责调控松材线虫头咽端特定组织的发育，三是参与调控轴突延伸及神经信号向突触后肌肉细胞的传导。.根据上述研究结果，我们认为madd-4基因在松材线虫的个体发育过程中表达部位相对稳定，并对肌肉系统的正常发育起着重要的调控作用，是保障松材线虫胚胎发育的重要基因。