粉红螺旋聚孢霉67-1产厚垣孢子相关基因的克隆及功能鉴定

Clonostachys rosea (=Gliocladium roseum) is a mycoparasite of many fungal plant pathogens and promising biocontrol agent. The development of chlamydospore preparation is of great significance to improve its stability and extend shelf-life. In this research, an effective strain 67-1 of C. rosea was chosen to screen key genes related to chlamydospore-production in the fungus basing on large amount of up-front research in the lab. The transcriptome during chlamydospore-forming process was achieved by manipulating liquid culture conditions under which chlamydospores and conidial spores were harvested, respectively. The cDNA expressing only in the isolate cultured in the medium for chlamydospore-generation and significantly up-regulated compared to those in Czapek Dox Agar which breeds conidia only were achieved by transcriptome sequencing analysis. Specific primers of the cDNA were designed and their expression levels in both conditions were quantified by using real-time PCR. The full-length sequences of differentially expressed genes were cloned based on the DNA sequence data of complete genome of 67-1, and their bioinformatics analysis was conducted. Gene knockout and gene complementation were conducted and the capabilities of chlamydospore-production of the mutants on the specific medium were determined to verify the function of the genes tested. Furthermore, some preliminary approaches will be done to explore the metabolic pathways in which the functional genes involved. This research will provide insights into the mechanism of chlamydospore formation and play an important role in improving fungal biocontrol agents.

粉红螺旋聚孢霉是多种植物病原真菌的寄生菌，具有良好的生防潜力，但目前所用的分生孢子制剂稳定性较差、不易贮存，因而限制了其应用。厚垣孢子制剂货架期长且生防效果稳定，但该类孢子不易大量稳定生产。迄今，对粉红螺旋聚孢霉产厚垣孢子相关基因的研究尚未见报道，研究厚垣孢子形成相关基因，探索厚垣孢子形成机理，对其生防应用具有重要意义。本项目以具自主知识产权的高效生防菌株67-1为材料，在已完成全基因组测序和获得厚垣孢子大量培养条件的基础上，拟对产分生孢子和厚垣孢子的菌体进行转录组测序分析，获得明显上调表达的cDNA，并进行实时荧光定量PCR验证；克隆获得相关基因全长；进行基因敲除和互补验证其功能，明确相关基因对67-1生成厚垣孢子的影响；初步探讨厚垣孢子形成相关的代谢途径。所得结果对揭示粉红螺旋聚孢霉厚垣孢子形成机理和研发稳定高效的生防真菌厚垣孢子制剂具有重要意义。

粉红螺旋聚孢霉是多种植物病原真菌的寄生菌，具有良好的生防潜力，但目前所用的分生孢子制剂稳定性较差、不易贮存，因而限制了其应用。厚垣孢子制剂货架期长且生防效果稳定，但该类孢子不易大量稳定生产。研究粉红螺旋聚孢霉厚垣孢子形成相关基因，探索厚垣孢子形成机理，对其生防应用具有重要意义。本项目以具自主知识产权的高效生防菌株67-1为材料，对其产厚垣孢子相关基因进行了克隆及功能鉴定。明确了氧胁迫对粉红螺旋聚孢霉产厚垣孢子的影响，为基因功能验证提供了新的检测方法；优化了遗传转化体系，转化率提高1倍以上。通过对67-1产分生孢子和厚垣孢子过程转录组测序分析，获得大量差异表达基因；通过荧光定量和软件分析筛选出SSD和ACT组合作为产孢相关基因研究的内参基因，获得了一系列产抗性孢子候选基因。对高差异表达的过氧化氢/过氧化物酶、聚酮合成酶、二氢吡啶二羧酸合成酶、己糖激酶等编码基因克隆全长，生物信息学分析，进而通过基因敲除和回补研究其对67-1产孢，以及对菌株生长和寄生能力的影响。明确了过氧化氢/过氧化物酶基因负调控粉红螺旋聚孢霉厚垣孢子的形成，通过添加作用底物可以对发酵产孢过程进行调控。研究未发现聚酮合酶基因对粉红螺旋聚孢霉生防能力有显著影响，但该基因可以明显抑制厚垣孢子的形成。二氢吡啶二羧酸合成酶基因可同时影响粉红螺旋聚孢霉产孢和寄生能力，基因敲除后厚垣孢子数量显著降低，回补后产孢能力恢复。项目期间在SCI及国内核心期刊上发表研究论文4篇，国际会议报告3次。相关研究结果对揭示粉红螺旋聚孢霉厚垣孢子形成机理和研发稳定高效的生防真菌厚垣孢子制剂具有重要意义。