东祁连山高寒草地牧草内生细菌的多样性及其与宿主互作对逆境胁迫的响应

The genomic DNA of endophytic bacteria isolated from dominant grass at alpine grassland in the Eastern Qilian Mountains of Tibet Plateau were will be extracted, amplified with 16S rRNA primers and then sequenced by high-throughput sequencing technology. The genetic diversity will be illustrated by analyzing the OTU species, OTU abundance, shannon index, alpha diversity index, beta diversity and bray coefficient. Simultaneously, total mRNA of endophytic bacteria will be extracted, enriched, and sequenced with high-throughput sequencing technology. To explore the functional gene diversity, sequences will be analyzed for functional gene annotation, functional classification, and gene abundance. After being treated with hotness, coldness and ultraviolet ray, the total mRNA of grass with or without endophytic bacteria will be extracted, purified, enriched and sequenced. To reveal responses of the interaction between endophytic bacteria and hosts to environmental stress (such as hotness, coldness or ultraviolet ray) at the gene level, the sequences will be analyzed for functional gene classification, annotation and comparison. Results of this project will reveal the genetic and gene diversity of endophytic bacteria from dominant grass at alpine grassland in the Eastern Qilian Mountains of Tibet Plateau, explore responses to environmental stress (such as hotness, coldness or ultraviolet ray) at the gene level, provide a basis for defining microbiology law and ecological service function in alpine grassland ecosystem, and lay the foundation to protect and utilize the microbial resources of alpine grassland.

本项目提取青藏高原东祁连山高寒草地优势牧草内生细菌的基因组DNA，扩增16S rDNA基因序列和高通量测序，分析OTU 物种、OTU 丰度、shannon指数、alpha多样性、beta多样性和bray系数等指标，明确其遗传多样性;同时，提取内生细菌的mRNA，纯化和富集，高通量测序后进行功能基因的注释及分类和丰度分析等，探明其功能基因多样性；高温、低温和紫外线等逆境胁迫带或不带内生细菌的牧草种群，后提取总mRNA，纯化、富集和高通量测序，进行功能基因的分类、注释和比较，揭示内生细菌与宿主互作对逆境胁迫在基因水平的响应。项目成果不仅可以明确青藏高原东祁连山高寒草地极端生境牧草内生细菌的遗传和功能基因多样性，而且在基因水平揭示内生细菌与宿主互作对逆境胁迫的响应，为阐明内生细菌在高寒草地生态系统正常演替中的微生物学规律和生态服务功能提供依据，也为高寒草地极端生境微生物资源的保护和利用奠定基础。

本项目利用传统技术与分子生物学手段相结合的试验方法，研究高寒草地牧草内生细菌多样性及生态服务功能，结果表明：（1）东祁连山高寒草地线叶嵩草和矮生嵩草内生细菌具有丰富的遗传多样性，且海拔和宿主种类影响丰富性。（2）带菌（E+）和不带菌（E-）紫花针茅逆境胁迫表明，内生细菌有助于宿主生化指标的稳定，提高宿主对逆境的忍耐能力; 在转录组水平高通量测序、比对分析和功能注释，高温胁迫下E+有1个基因上调表达，其为植物防御过程中具有重要作用的Jacalin类凝集素，3个下调表达；低温胁迫下E+有421个上调表达，152个下调表达；紫外线胁迫下E+有3837个上调表达，2870个下调表达。（3）内生细菌具有良好的抑菌、溶磷、固氮及产IAA功能，功能多样性丰富；明确了优良拮抗细菌的抑菌机制、定殖动态、防病促生作用及对土著微生物的影响，研制了微生物菌剂并进行了小试。（4）内生细菌ZA1的次级代谢产物对酸、碱、温度和UV稳定，且同时产生挥发性抑菌代谢物、小分子抑菌化合物和脂质抑菌物质，也有促进植物生长的化合物(具体种类见附件)。项目研究结果不仅明确青藏高原高寒草地极端生境牧草内生细菌具有丰富的遗传多样性和功能多样性，而且揭示在逆境胁迫下内生细菌影响宿主基因表达，有助于提高其抵抗逆境能力，为阐明内生细菌在高寒草地生态系统中的生态服务功能提供依据，也为极端生境微生物资源的保护和利用奠定基础。