霾对艾叶免疫功能与叶际菌群的干扰模式研究

The haze as a popular air pollution is another important factor that attenuates the quality of Chinese herbs, yet the damage mechanisms are waiting for further exploration. In the present proposal, artemisia (Artemisia Princeps Pamp. Var.) will be recruited as the representation of Chinese herbs to probe the damage pattern of haze on the antibacterial immunity of artemisia and their phyllospere microbe. Artemisia samples living in the middle haze condition (the air quality index (AQI) 201-250 will presented for 80-100 d per year), and series samples of artemisia lived in the natural light climatic chamber, where the AQI will be regulated among 201 to 250, for 1w, 2w, 4w and 8w. Every sample will be analyzed as follow. (1). Check the chlorophyll strength and necrosis condition in quo，observation of the micrography and callose formation by the pathological slices of leaves. (2). Analysis of the construction and function of the phyllospere microbiota by resequencing of the metagenomics and real-time PCR. (3). Observation of amount and distribution of FLS2 and Flg22, the pattern recognition receptors (PRRs) of pathogens in plant leaves, by immunohistochemistry, and the activities of BAK1/SERK3 by ELISA. (4).Detect of the signal transportation pathways triggered by SA-JA-ET cascades, the plant hormones that are essential in the regulation of plant growth, antibacterial immunity and survival, by the approach of microchip and LC/GC-MS. (5). Quantification of the antibacterial compounds such as 5-tert-Butylpyrogallol within the artemisia leaves by HS-SPME-GC-MS, and their antibacterial effects by smoking methods. The above researches will explore the multi-level and dynamic pattern of the antibacterial immunity and phyllospere interfered by haze, and found a brand-new theory that haze impacts the quality of certain Chinese herbs.

霾等空气污染是造成中药材品质下滑的又一重要因素，但其病理机制缺乏研究。本课题以叶类中药艾药典品种为代表，探讨霾损伤艾叶抗菌免疫功能、叶际菌群及艾叶抗菌药效成分积累的病理机制。拟纳入中重度霾环境自然生长与模拟霾气候室内生长1w,2w,4w,8w之艾叶，测定叶绿素荧光强度、坏死斑，观察微地形、胼胝质等叶面病理改变；高通量测序结合定量PCR分析叶面菌群结构与功能；免疫组化法检测病原菌模式识别受体FLS2,flg22含量与亚细胞分布模式，ELISA法测定其效应酶BAK1/SERK3活性；信号通路芯片法分析免疫相关植物激素SA-JA/ET激发的信号通路网络基因表达水平，LC/GC-MS测定相应激素含量；HS-SPME-GC-MS联用测定5-叔丁基连苯三酚等艾叶抗菌药效成分含量，烟熏法检测其空气污染菌拮抗能力。多层次、动态研究霾危害艾叶－叶际菌群共生体的分子病理学机制，创立霾影响叶类中药品质的新学说。

霾对艾叶等药用植物的影响机制复杂，但缺乏研究。本课题拟以与霾直接接触的叶际菌群为研究切入点，比较分析霾环境及洁净对照地区的艾叶-叶际菌群的结构与功能差异，探讨霾对艾叶-叶际菌群的干扰机制。分别采集来自代表性霾环境和相对洁净区的成熟艾叶叶片，提取艾叶表面微生物总DNA，Illumina HiSeq高通量平台进行测序，探讨霾环境对艾叶叶际菌群的干扰机制。结果发现：.雾霾组的基因数目少于清洁组，叶际菌群多样性降低。通过比较分析不同样品间的基因共有、特有信息,发现雾霾组的基因数目为652,138,清洁组的基因数目为675,468,两者共有的基因数目为513,460。提示雾霾组叶际菌群的基因数目少于清洁组，叶际菌群多样性降低。经过spearman 相关系数分析后得出,两者基因丰度模式的相似度差异大。霾对艾叶叶际菌群的结构干扰较大。在物种注释中，经主成分分析、无度量多维标定法、主坐标分析等降维分析结果，雾霾组和清洁组两组之间距离较远，提示两组之间叶际菌群结构差异较大。在物种注释中，Bray-Curtis 距离矩阵样品间门种类聚类分析结果显示，雾霾组与清洁组叶际菌群差异显著,聚类距离较远。霾对艾叶叶际菌群的新陈代谢等功能产生显著影响。 经三种数据库：KEGG、eggNOG以及 CAZy的对比得出，在KEGG数据库里新陈代谢方面的功能基因数目被注释的最多，约占36%。在eggNOG数据库中则是在已知功能基因注释数目中约占12%的复制、重组及修复方面的功能占的比例最高，在CAZy数据库里，已知功能的基因被注释最多的是糖苷水解酶方面的功能。功能注释聚类热图显示出，叶际菌群功能在雾霾组和清洁组间有显著差异。在功能注释中，雾霾组和清洁组两组之间距离较远，提示两组之间叶际菌群功能差异较大。雾霾组叶际菌群真菌的丰度降低,细菌的丰度升高. 在门水平上,其中丰度前十的微生物门种类丰度占据了98%以上的百分比,且雾霾组与清洁组在门水平上排名前十的微生物菌群相同,两者仅有占比差异。雾霾组与清洁组相比较,雾霾组的变形菌门、放线菌门、异常球菌-栖热菌门丰度显著升高,而子囊菌门、担子菌门丰度均显著降低。总体而言,雾霾组较清洁组,真菌的丰度降低,细菌的丰度升高。霾环境导致艾叶叶际菌群在结构和功能上发生变化，主要体现在初级代谢产物及次级代谢产物的明显减少，另外艾叶通过活跃基因的复制、重组与修复功能，以及抑制R