马铃薯晚疫病菌Phytophthora infestans中miRNA的研究与分析

Late blight, caused by Phytophthora infestans (Mont.) de Bary (Oomycota, Stramenopiles) is the most important diseases threatening potato production in many countries, especially in China. This plant pathogen causes billions of lost in potatoes, tomatoes and other members of the Solanaceae worldwide. In the life cycle of P. infestans, sporangia germination is a critical stage in the process of P. infestans infection. The genome of P. infestans, completed sequencing in 2009, was found to be considerably larger (240 Mbp) than that of most other Phytophthora species whose genomes have been sequenced; Phytophthora sojae has a 95 Mbp genome and Phytophthora ramorum had a 65 Mbp genome. It also contained a diverse variety of transposons and many gene families encoding for effector proteins that are involved in causing pathogenicity. However, MicroRNA is rarely researched in this pathogen. MicroRNAs are small non-coding RNAs of approximately 22 nt with important regulatory roles in post-transcriptional regulation of gene expression. Most published documents indicated that miRNAs play important roles in plant development, such as organ separation, leaf development and polarity, lateral root formation, floral organ identity and reproduction, etc. MiRNAs are alsovery important in regulation of animal development, differentiation, carcinogenesis, and immunoreaction. Since the discovery of the first miRNA in C. elegans, miRNAs have been widely found in animals, plants, and algae, but not in P. infestans or Oomycetes. Our preliminary work shows that there is a highly chance that miRNA also exist in P. infestans and functions to their target genes. This project intends to get the small RNA sequencing data by Illumina platform with the three different developmental stages tissues, non-germinated sporangia, germinated sporangia and mycelium. The sequencing data will be used to analyze the potential miRNAs and predict their hairpin precursors and target genes. Small RNA hybridization will be performed to confirm the presence of miRNAs. Differential expression profiling among different tissues will be built through Bioinformatics analysis. We are going to identify the differentially expressed miRNAs in sporangium germination process, test the expression level with qPCR and predict their target genes. The mechanism of miRNA worked with its target genes will also be researched in this project.

马铃薯晚疫病作为马铃薯生产中的头号病害，是引起粮食作物产量损失最严重的一种真菌病害。孢子囊萌发是P. infestans侵染过程中至关重要的阶段。miRNA在动植物中广泛存在，可通过识别靶基因实现转录后的基因调控。本项目研究拟选取P. infestans菌株三个不同发育时期的组织样品- - 未萌发的孢子囊、萌发的孢子囊和菌丝，利用Illumina技术对三个样本进行小RNA测序，通过数据分析预测P. infestans不同发育阶段的milRNA及其作用的靶基因。并通过sRNA杂交实验证实部分milRNA的存在，找到其发夹前体，初步研究milRNA对靶基因作用机制。通过构建样品之间的小RNA差异表达谱，找出在孢子囊萌发过程中特意表达的miRNA及其靶基因，为深入研究P. infestans与植物寄主的相互作用机制奠定基础。

马铃薯是世界粮食作物中仅次于水稻和小麦的第三大作物。马铃薯晚疫病是马铃薯生产中的头号病害，是所有引起粮食作物产量损失的病害中最严重的一种卵菌病害。本项目目标为研究和分析卵菌植物致病菌P. infestans中的miRNA。通过项目研究，证实卵菌植物致病菌P. infestans中确实存在miRNAs，检出与其它物种同源的保守miRNA32个，新预测候选miRNA 2098个，并分析了miRNA序列特征，发现其5’端首位具有碱基U的偏好性。保守miRNA和新预测miRNA的分布长度峰值是21nt(19.66%)，25nt(33.74%)。small RNA blot实验显示部分测序获得的miRNA序列能够在P. infestans中杂交到。.构建不同时期样品miRNA的差异表达谱，其中未萌发孢子囊S与菌丝H相比共有256个miRNA显著差异表达；未萌发孢子囊S与萌发孢子囊GS相比共有213个miRNA显著差异表达；菌丝H与萌发孢子囊GS样品相比共有125个miRNA显著差异表达。通过qPCR检测不同时期样品中miRNA表达量，部分miRNA表达量与测序结果相符。进一步预测差异表达miRNA的靶基因，通过 GO的富集性分析确定差异表达基因行使的主要生物学功能。.实验筛选了大量miRNA和预测的靶基因进行实验验证，最终利用5’RACE鉴定出在萌发孢子囊和菌丝样品中，let-7对其预测靶基因PIK-A mRNA具有切割作用，切割作用位点在let-7与PIK-A mRNA结合位点的10、11位和11、12位之间，其中大部分切割位点发生在10与11位之间（14/16）。而在未萌发孢子囊样品中并未检测到切割作用，这与let-7在未萌发孢子囊样品中表达量最低的结果相符。该结果表明在卵菌P. infestans中也存在通过切割靶标mRNA调控靶基因表达的方式，且在不同组织样品中有差异，预示着卵菌中的miRNA可能与植物和动物中的miRNA一样，通过调控靶基因表达参与生长发育等的调节途径。同时，该项实验结果也表明这种miRNA调控靶基因的模式是比较保守的，不仅在高等动植物中存在，也在一些真核微生物中得到进化。.本项目通过对P. infestans miRNA的研究和分析，推动了了对卵菌分子遗传学的研究，有助于认识和深入研究P. infestans与植物寄主的相互作用机制.