野生越橘杜鹃花类菌根真菌定殖关键基因筛选及定殖机理初探

In this project, ericoid mycorrhizal fungi are isolated and identified from wild Vaccinium in northeast of China, and the community structure characteristic will be analyzed. In the process of invasion of ericoid mycorrhizal fungi, the dynamic characteristics of the enzymes from both fungi and plant are studied by the immuno-fluorescent method, so we will wise up the interaction mechanism between the dynamic characteristics of these enzymes and inoculation of fungi. In the process of invasion of ericoid mycorrhizal fungi, the variety of some substance from secondary metabolism in root is analyzed and identified, and we will make an attempt to find the function of the substance from secondary metabolism. Via analysis on several factors, such as environment, signal molecular, we will quest for the influence of colonization of mycorrhizal fungi when the factors perform a combined-action. Based on the high-throughput sequencing of transcription and laser microdissectin, we will study the diversity of transcription among no-inoculation cells, inoculation cells with mycelium, and inoculation cells lacking mycelium, so we can understand the change for symbiosis-related genes in root of Vaccinium in the process of invasion of ericoid mycorrhizal fungi. In addition, proteinic change will be also studied by the method of isobaric tags for relative and absolute quantitation, and the relativity between the result of transcription and proteome is analyzed. Several genes, which are obtained by analysis on transcription and proteome, are located with green fluorescent protein gene tag in cells. Combined with laser microdissectin, the dynamic characteristics of symbiosis-related genes in the invasion process of ericoid mycorrhizal fungi will be determined with real time PCR and in situ hybridization. Lastly, the influence of symbiosis-related gene silence on colonization of mycorrhizal fungi is analyzed by RNAi technique on the basis of the dynamic characteristics of symbiosis-related genes.

对东北地区野生越橘杜鹃花类菌根真菌进行分离、鉴定。利用免疫荧光技术，分析菌根菌定殖过程中产酶及植物防御酶动态变化，了解其与接种菌根菌间相互作用机制。从环境因子、信号分子等方面对菌根菌定殖的影响因子进行分析，分析不同因子协同作用对菌根菌定殖的影响。结合显微切割技术，基于微量细胞高通量转录组测序技术分析未接种菌根细胞、接种带/无菌丝细胞间的转录组差异，筛选越橘菌根共生相关差异表达基因。利用iTRAQ技术分析菌根菌侵入后菌根蛋白质的变化，并分析其与转录组变化的相关性。基于转录组、蛋白质组分析中获得的差异表达基因，利用绿色荧光蛋白标记对其进行亚细胞定位。结合显微切割技术，利用Real time PCR、原位杂交技术分析差异表达基因的时空变化动态；在此基础上，利用RNAi技术分析相关基因表达受抑制对菌根菌定殖的影响。

越橘(Vaccinium)是最重要的新兴小果类果树之一，世界分布广泛。自然条件下几乎所有的越橘细根都有内生菌根真菌的寄生，其克服了越橘由于没有根毛而造成的对水分及营养的吸收困难，从而改善植株营养状况，增强植株的抗逆性，提高越橘的产量。尽管越橘育苗技术大力发展，但大规模种植之后成活率相对较低，进入稳产期较慢，这是困扰当前越橘扩大规模生产的主要瓶颈问题。尽管中国具有丰富的越橘菌根真菌资源，但实验室分离的菌根真菌常常不能够有效地定殖于田间，其主要原因在于当前对越橘菌根共生的分子机制尚不了解。本项目从转录组、蛋白质组、microRNA等方面探索分析越橘菌根共生相关影响因子，为揭示杜鹃花类菌根共生机理奠定了重要的理论基础。. 本项目对东北地区越橘杜鹃花类菌根真菌进行了分离、鉴定，获得了大量杜鹃花类菌根真菌。解磷能力分析显示较多菌根真菌菌株具有解磷能力。获得了绿色荧光蛋白标记的菌根菌，可有效分析菌根真菌的侵入动态。对菌根真菌的细胞壁降解酶和漆酶活性进行了测定，显示越橘杜鹃花类菌根真菌可产生细胞壁降解酶和漆酶。推测菌根真菌可能通过细胞壁降解酶破坏植物细胞壁组织，以利于真菌侵入；之后植物可能会产生酚类物质进行防御，而菌根真菌通过产生漆酶可以将酚类物质氧化，从而促进菌根真菌的侵入及定殖。基于荧光标记的特异性抗体，利用免疫荧光技术可有效分析菌根菌在越橘根部分布。利用转录组测序技术分析了未接种/接种菌根组织间的转录组差异，获得了大量越橘菌根共生相关差异表达基因。利用 iTRAQ 技术分析菌根菌侵入后菌根蛋白质的变化。研究中还对越橘植株中的microRNA进行了挖掘，并对部分microRNA进行了Stem-loop PCR验证。获得了接种/未接种菌根真菌越橘植株中差异microRNA，经靶标基因预测，一些差异microRNA靶标基因与转录组测序中获得的差异表达基因相似。