粗糙脉孢菌NC-3降解水稻秸秆的作用机理研究

For ecological protection and agricultural sustainable development, scientific and rational utilization of straw resources is an important task. Implementation of straw degradation in field by natural microbes can effectively promote the utilization of straw in China. Our previous study showed that the degradation rate of rice straw significantly increased more than 30% by inoculated with Neurospora crassa (NC-3). However, the degradation mechanism of rice straw by NC-3 is still not clear. This project plans to: (1) Under the condition of different temperature, different time and different fermentation formula, determination of the cellulase activity of NC-3, and comparison between the control group and the treatment group. (2) Through the technology of ordinary microscope, scanning electron microscope and microbiology analysis, to elucidate the suitable carbon nitrogen ratio of NC-3 on spore germination, mycelial growth, root colonization and enzyme production, and preparation of the solubble powder for NC-3. (3) Using two-dimensional electrophoresis of protein, mass spectrometry identification, transcriptome sequencing and fluorescence quantitative PCR, combining with bioinformatics analysis to explain the molecular mechanism of rice straw degradation by NC-3. The results will establish the theoretical foundations, such as preparation high efficient straw degrading microbial agents, application the technologies of straw degradation in field, soil improvement, reduction of fertilizer and so on.

科学合理利用秸秆生物质资源是我国生态保护和农业可持续发展的重要任务，实现田间原位秸秆微生物快速转化降解，能有效促进秸秆资源的还田利用。本项目组从油菜—水稻轮作土壤分离获得粗糙脉孢菌（Neurospora crassa NC-3），可使水稻秸秆腐解率提高30%以上。本研究拟通过不同温度、时间和发酵配方，测定NC-3的纤维素酶活性，比较对照组与处理组之间的差异；应用普通光学显微镜、扫描电子显微镜以及常规的微生物学分析，确定适合NC-3孢子萌发、菌丝生长、定殖和产酶的碳氮比，同时研制NC-3可溶性粉剂；通过NC-3的蛋白质双向电泳及斑点质谱鉴定、转录组测序、荧光定量PCR验证差异基因和蛋白的表达，结合生物信息学分析，揭示NC-3降解水稻秸秆的分子作用机理。本项目的开展，将为进一步研究作物秸秆原位降解还田、改良土壤、节肥减施等技术的研发和应用奠定理论基础。

科学合理利用秸秆生物质资源是我国生态保护和农业可持续发展的重要措施，实现田间原位秸秆微生物快速转化降解，能有效促进秸秆资源的还田利用。项目组从油菜—水稻轮作土壤分离获得的粗糙脉孢菌NC-3，可使水稻秸秆降解率提高30%以上。本研究通过室内模拟和盆栽试验，探讨了NC-3在水稻秸秆上的定殖规律、对水稻秸秆降解以及后茬油菜幼苗的影响；通过转录组测序结合生物信息学分析，探讨了NC-3降解水稻秸秆的分子作用机理。结果表明：①水稻秸秆接种粗糙脉孢菌NC-3，12h后孢子开始萌发并逐步生长，48h后秸秆正面长满菌丝且有少量菌丝穿透秸秆在反面生长，72h后秸秆正反面菌丝生长均达最大值。②NC-3孢子萌发的最适碳氮比为18~23：1。③相比无菌水处理，水稻秸秆接种NC-3培养7d、14d和21d，秸秆降解率分别提高2.27、7.30和3.18个百分点；秸秆纤维素、木质素以及总酚酸含量分别下降2.03、10.67、10.45个百分点，0.72、0.88、1.33个百分点和7.6%、6.9%、6.4%，处理间差异显著。NC-3对水稻秸秆、纤维素、木质素和总酚酸的降解作用主要发生在秸秆还田的前两周（0~14d），对半纤维素的降解作用在培养14d后逐渐增强。水稻秸秆还田会显著降低油菜发芽率和成苗率，且随秸秆还田量的增加抑制作用增强，但接种NC-3能显著提高油菜发芽率和成苗率（相比无菌水，分别提高3.33和9.67个百分点）。④基因本体（GO）功能注释和通路富集（KEGG）分析表明，NC-3主要通过半乳糖代谢、果糖和甘露糖代谢有效提高水稻秸秆降解效率。综上所述，粗糙脉孢菌NC-3能在水稻秸秆上快速定殖，能有效加快水稻秸秆降解及酚酸类物质转化，显著提高后茬油菜发芽率和成苗率。研究结果将为进一步研究作物秸秆原位降解还田、改良土壤、节肥减施等技术的研发和应用奠定理论基础。