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苹果褐斑病是由苹果褐斑病菌(Diplocarpon mali)引起的苹果(Malus domestica)早期落叶病害之一,该病害引起叶片黄化和早落,造成严重的经济损失。本研究采用新一代高通量测序技术Illumina Hiseq2500对被苹果褐斑病菌侵染的山定子(M.baccata)叶片进行转录组测序,利用生物信息学方法对基因表达和功能进行研究。通过测序获得46.86 Gb碱基序列信息,对测序数据进行序列过滤、拼接、组装、去冗余,共获得48 868个单基因簇(unigenes)。从长度分布、GC含量、表达水平等方面对unigenes进行评估,结果显示,测序质量好,可信度高。此外利用美国国家生物技术信息中心(National Center of Biotechnology Information,NCBI)的NR(Non-Redundant)、Swiss-Prot蛋白质序列数据库(Swiss-port Protein Sequence Database)、KOG(Eukaryotic Orthologous Groups of Proteins)、基因本体论(Gene Ontology,GO)、京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)等常用数据库对组装的48 868条unigenes进行同源性预测,NR数据库中的序列同源性比较表明,25 797个unigenes直接比对到了蔷薇科苹果基因上。将unigenes与KOG数据库进行比对,根据其功能大致可分为25类。以KEGG数据库作为参考,依据代谢途径可将unigenes定位到145个代谢途径分支,包括核糖体代谢通路、碳水化合物代谢等。利用RSEM软件进行差异表达计算,edge R软件进行差异表达分析,通过设定阈值错误发现率(false discovery rate,FDR)<0.05,log2(差异倍数(fold change,FC))>2或log2(FC)<-2筛选差异基因,共鉴定出上调基因1 230个,下调基因1 869个,KEGG Pathway富集结果表明,有94个差异基因参与酪氨酸代谢、植物病原体相互作用、植物激素信号转导以及氨基酸的生物合成等相关通路。q RT-PCR验证部分上调基因结果与转录组分析结果一致。本研究通过二代高通量转录组测序技术研究山定子在抗褐斑病中的相关基因,为明确山定子抗褐斑病的分子机理提供了基础资料。
Apple brown spot is one of the early deciduous diseases of apple (Malus domestica) caused by Diplocarpon mali, which causes yellowing and catharsis of the leaves, causing serious economic losses. In this study, a new generation of high-throughput sequencing technology Illumina Hiseq2500 was used to transcribe the leaves of M. baccata infected with P. moruciferum, and the bioinformatics methods were used to study the gene expression and function. A total of 48 868 unigenes were obtained by sequencing the 46.86 Gb nucleotide sequences and sequencing, splicing, assembling and de-redundant sequencing. The unigenes were evaluated in terms of length distribution, GC content and expression level. The results showed that the sequencing quality was good and the reliability was high. In addition, NR (Non-Redundant), Swiss-port Protein Sequence Database, KOG (Eukaryotic Orthologous Groups of Proteins) of the National Center for Biotechnology Information (NCBI) Homology prediction was performed on 48,868 unigenes assembled by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and comparison of sequence homology in NR database Showed that 25,797 unigenes were directly aligned to Rosaceae apple genes. The unigenes and KOG database for comparison, according to its function can be broadly divided into 25 categories. Based on the KEGG database, unigenes can be mapped to 145 metabolic pathways based on metabolic pathways, including ribosomal metabolic pathways, carbohydrate metabolism and the like. Differential expression analysis was performed using RSEM software. Edge R software performed differential expression analysis by setting threshold false detection rate (FDR) <0.05, log2 (fold change, FC)> 2 or log2 (FC) ) <- 2, a total of 1 230 genes were up-regulated and 1 869 genes were down-regulated. The KEGG Pathway enrichment results showed that 94 different genes were involved in tyrosine metabolism, plant pathogen interactions, phytohormone signal transduction Guide and amino acid biosynthesis and other related pathways. q RT-PCR validation part of the up-regulated gene results and transcriptome analysis results. In this study, the second generation of high-throughput transcriptome sequencing technique was used to study the relative genes of Rhododendron simvastatin in resistance to brown spot disease, which provided the basic information for clarifying the molecular mechanism of Rhizoctonia solani.