微流控芯片质谱联用在植物内生菌培养组学研究中的应用

Plant endophytes have tremendous species which are great treasure of medicinal resources. In addition to the special way of life, the active substances isolated from the plant endophytes are structurally diverse and have low toxicity. The agar streak plate method of plant endophytes is laborious. Many rare species cannot obtain because it is difficult to observe and collect small colonies. The low efficient screening methods such as the inhibition zone test also restrict the discovery and development of new active substances. To overcome these defects, we try to introduce the culturomics to study the plant endophytes based on our previous microfluidic chip mass spectrometry studies. Nanoliter microfluidic droplets will be used as a culture medium of endophytes to obtain the mass culturome in different culture conditions. By using the 16S rDNA PCR amplification and sequencing and microfluidic mass spectrometry, we will establish the high throughput, rapid identification of the culturable endophytes in culturome. By quantitative analyzing of the mass data, we will select the stains to establish the stains library. Finally, the fluorescence labeled stains such as methicillin resistant staphylococcus aureus would be used as the indicator stain to screen the endophytes in the stains library by nanoliter micro droplet fusion technology and the gradual enlargement of co-culture, the stains with strong antibacterial activity will be selected for the subsequent discovery of new products.

植物内生菌种类丰富，蕴藏量大，是有巨大开发潜力的药用资源宝库。加之其特殊的生存方式，从植物内生菌中分离得到的活性物质结构多样，且毒性较低，相比化学合成药物具有明显优势。目前植物内生菌纯培养的琼脂平板划线分离技术通量低，无法实现微小菌落的观察和收集，许多稀有物种难以获得；而抑菌圈等筛选方法的低效率也阻碍了新型活性物质的发现和开发。为了克服这些缺陷，本项目拟从最近兴起的微生物培养组学角度出发，将纳升微流控液滴作为植物内生菌的培养器进行单细胞纯培养，获得大量培养条件下的培养组。利用扩增子测序和微流控芯片质谱联用，对植物内生菌培养组进行高通量快速鉴定，通过数据定量分析挑选目标菌株建立菌株文库。最后以带荧光标记的模式菌为指示菌株，通过微液滴融合、逐级放大共培养的方法，对菌株文库中的植物内生菌进行高通量筛选，挑选出具有强抑菌活性的菌株，为后续新型天然产物的发掘奠定基础。

植物内生菌是目前有待开发的一类非常重要的微生物资源，是发现新的生物活性分子以及阐明内生细菌与宿主相互作用机制的关键因素。但是目前传统平板培养分离的研究方法具有通量低、试剂消耗大等缺陷，只能培养出约1%的内生菌。微液滴具有体积小，消耗低，通量高等优势，是理想的细菌培养器。而MALDI-TOF质谱作为一项新的微生物鉴定技术，具有简单快速的特点。本课题首先对杨树内生菌进行较全面的组学研究，比较了微流控液滴培养和96孔板高通量分离鉴定对于杨树内生菌分离培养的优缺点，获得了杨树内生菌菌株资源；然后优化植物内生菌质谱分析的预处理方法，实现了基于MALDI-TOF的植物内生菌快速鉴定，建立了杨树内生菌的质谱鉴定数据库；最后开发了基于荧光的高通量的微液滴菌株筛选技术，提高了菌株分离培养的效率，克服了传统皮纳升液滴微流控在进行分析后，无法回收极微量目标菌株的难题，为活性产物的发掘奠定基础。另外本项目获得的杨树内生菌株资源为后续代谢产物分析、细菌与杨树之间的互作机制研究等具有重要意义。