松材线虫性比调节的种间竞争策略及分子机制研究

Interspecific competition occurs fiercely between Bursaphelenchus xylophilus and Bursaphelenchus mucronatus. Our previous studies showed that the female ratio increased in both B. xylophilus and B. mucronatus when they were mixed and cultured together in the laboratory. However, the female ratio of B. xylophilus increased more efficiently than that of B. mucronatus, leading to its more efficient population propagation and enhancement of interspecific competitiveness. Based on these findings, this project will quantitatively analyze the sex ratio change of B. xylophilus in mixed culture with B. mucronatus, demonstrate the crucial role of sex ratio in fighting off rival, and elucidate the competitive strategy of B. xylophilus against B. mucronatus by increasing female ratio. The karyotype of B. mucronatus will be determined by G-band and DAPI staining analysis. The key sex determining genes will be predicted and confirmed in term of comparative genomics with the high-throughput sequencing data, which were obtained in our previous studies. The spacial-temporal expression pattern of key genes will be investigated by qPCR, mRNA in situ and transgenic microinjection, and their crucial roles in sexual development will be defined by RNA interference. In addition, DNA methylation and alternative splicing of selected key genes will be analyzed by epigenetic sequencing and RNA-seq to reveal the regulation process, which will shed light on the molecular basis of sex-ratio change in the process of interspecific competition. The results of this project will not only reveal the competitive strategy, but also gain insight into the underlying molecular basis of sex determination and differentiation of B. xylophilus, as well as provide the theoretical principles to design specific agents against pine wilt disease in future.

在松材线虫病扩散过程中，松材线虫和拟松材线虫存在激烈的种间竞争。前期研究发现，在混合培养条件下两种线虫的雌雄性比都有明显的变化，但松材线虫能够更加有效地提高雌虫的比例，增强自身的种群繁殖能力和种间竞争能力。本项目拟在前期研究的基础上，定量分析松材线虫的性比调节能力，明确性比调节在种间竞争过程中的关键作用，阐明松材线虫性比调节的种间竞争策略；采用G-band和DAPI染色技术分析松材线虫的染色体，明确其性别决定的遗传学基础；挖掘松材线虫的性别决定相关基因，研究关键基因对性别分化的决定作用；分析DNA甲基化状态和选择性剪接对性别分化的影响，阐明种间竞争对松材线虫性别分化的调控作用，揭示松材线虫性比调节的种间竞争分子机制。本研究结果不仅能够阐明松材线虫的种间竞争策略，丰富入侵生物学内容，也能够揭示松材线虫性别分化的分子机制，为进一步开展松材线虫的发育生物学研究和设计特异性的病害防控措施提供思路。

在松材线虫病扩散过程中，松材线虫和拟松材线虫存在激烈的种间竞争。前期研究发现，在混合培养条件下两种线虫的雌雄性比都有明显的变化，但松材线虫能够更加有效地提高雌虫的比例，增强自身的种群繁殖能力和种间竞争能力。本项课题通过线虫种群数量分析、比较基因组学和转录组学和分子生物学手段，系统开展了以下几个方面的研究：（1）研究了松材线虫性比调节的种间竞争策略，明确了松材线虫通过提高雌雄比增强自身的雌性数量，并通过交配干扰剥夺本土拟松材线虫的交配机会，获得种间竞争优势，最终成功替代拟松材线虫并占据生态位；（2）挖掘并成功克隆了17个松材线虫性别决定基因，验证了关键性别决定基因的生物学功能，明确了松材线虫性别决定的调控通路，为系统揭示松材线虫性别决定与分化的分子机制垫定了坚实的基础；（3）初步分析了松材线虫性别决定与分化对温度的响应，明确了松材线虫性别分化对温度响应的关键窗口期，并开展转录组研究，为进一步深入分析其性别决定的生物学过程提供基础。该项目在行为学研究的基础上，阐明了松材线虫性比调节的种间竞争策略，丰富了入侵生物学的内容，初步揭示了松材线虫性别决定与分化的分子基础，具有重要的科学价值。

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