越橘microRNA分子挖掘及越橘与杜鹃花类菌根真菌互作中的microRNA解析

MicroRNAs (miRNAs) are unearthed by high-throughout sequencing and bioinformatic analysis from different tissues of Vaccinium, and validated by molecular hybridization with gene chip. The difference of miRNA expression among no-inoculated tissues and inoculated tissues of Vaccinium by ericoid mycorrhizal fungi, inoculated cells with mycelium, and inoculated cells without mycelium, are analyzed by high-throughout sequencing. Combining enzymatic probing, nuclear magnetic resonance spectroscopy with bioinformatic analysis, the tertiary structures of complex of miRNA-target gene mRNA 3′UTR, miRNA and pre-miRNA related to colonization of ericoid mycorrhizal fungi, are detemined. Via analyzing the effect on tertiary structures and the colonization of ericoid mycorrhizal fungi by different styles of miRNA variation, we will understand the relativity between the structures and function of miRNA. The function of miRNA is studied using overexpression of miRNA, mutant techniques and other methods, and the influence of mycorrhizal colonization and the interaction among miRNA, precursor and target gene related to colonization of ericoid mycorrhizal fungi are also analyzed. Via screening the miRNAs and studying the relativity between tertiary structures and function of miRNA, it is likely to understand in depth the mechanism of colonization of ericoid mycorrhizal fungi of Vaccinium via a novel perspective of miRNA.

运用高通量测序及生物信息学技术挖掘越橘microRNA(miRNA)，并进行基因芯片杂交验证。通过高通量测序技术分析菌根真菌不同侵染阶段未接种/接种真菌的越橘组织、接种菌根真菌带/无菌丝根组织细胞miRNA的表达差异。结合RNA酶学标记、核磁共振波谱法及生物信息学等方法，分析菌根真菌定殖相关的miRNA、pre-miRNA、miRNA－靶基因mRNA 3′UTR复合体的三级结构。探讨不同miRNA变异模式对RNA三级结构及菌根真菌定殖的影响，了解miRNA结构与其功能的相关性。利用miRNA过表达及突变等策略对miRNA功能进行分析，了解菌根真菌定殖相关miRNA及靶基因、pre-miRNA的相互作用及其对菌根真菌定殖的影响。通过筛选越橘菌根真菌定殖相关miRNA并分析其三级结构与功能相关性，可以从全新的miRNA视角深层次地剖析越橘杜鹃花类菌根真菌的定殖机理。

MicroRNA(简称miRNA)对植物生长发育起着重要调控作用。作为菌根中的一种重要类型，杜鹃花类菌根共生相关miRNA尚未见报道。本项目利用高通量测序技术挖掘越橘miRNA分子，筛选杜鹃花类菌根真菌(Ericoid mycorrhizal fungi, EMF)定殖相关的miRNA，并分析其功能及作用机制。主要研究结果如下：. ①本项目定期采集野生越橘样品，进行菌种分离，获得了大量EMF、深色有隔内生真菌(Dark septate endophytes, DSE)菌株。发现EMF与DSE定殖呈负相关，提出了二者在越橘毛根中假定的共定殖模式。建立了利用原位PCR、绿色荧光蛋白基因标记技术监测菌根真菌定殖的技术体系。. ②对越橘组织样品构建了小RNA文库，进行了高通量测序，获得已知miRNA 282个；预测出新miRNA 100余个。分别利用Expdiff法、荧光定量PCR筛选不同接种处理差异表达miRNA。利用降解组测序分析miRNA的靶基因，结合其特点，初步筛选出了一些菌根真菌定殖相关miRNA。建立了基于单细胞-荧光定量PCR技术检测miRNA的技术体系。发现有菌丝细胞中，一些miRNA的表达量高于无菌丝细胞。. ③筛选差异表达miRNA的靶基因Unigene，对7个miRNA靶基因进行了过表达及沉默载体构建。利用PCR技术获得9种miRNA的pre-miRNA序列。经全基因合成、体外转录获得了miRNA-靶基因mRNA单聚体RNA及变异体，可用于分析三级结构及变异对三级结构的影响。对pre-miRNA进行了三级结构预测，一些破坏三级结构的变异可能影响pre-miRNA与酶的结合，影响miRNA产生及生物功能的发挥。基于RNA三级结构预测，分析了miRNA种子区序列/结构变异对其与靶基因mRNA结合的影响。构建了miR157a-5p、miR535d过表达及沉默载体，过表达、沉默载体转化分别提高、降低了miRNA表达量；高表达植株的抗逆相关生理活性物质活性升高，并提高了菌根形成效率。