苹果褐斑病菌侵染苹果属山定子的转录组学分析

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摘要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，edgeR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。qRT-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。

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	李海录
	宋艳艳
	冯浩
	黄丽丽
	强晓玉
	韩青梅

关键词 ：山定子, 苹果褐斑病, 转录组, 高通量测序

Abstract：Marssonina leaf blotch of apple caused by Diplocarpon mali, is one of the main apple (Malus domestica) leaf defoliation diseases in apple production areas, which can result in severe etiolation and abscission of leaves during the growing season. In this study, a new generation of high-throughput sequencing technology Illumina Hiseq2500 transcriptome sequencing was used for transcriptome sequencing in M. baccata infected with Diplocarpon mali, and then bioinformatic methods were used for gene expression profile and gene function prediction. The results showed that 46.86 Gb sequence information was abtained. Through filtering, splicing, assembling and going redundancy, 48 868 unigenes were abtained. After assessing the length distribution, GC content, expression level and other aspects of the unigenes, the sequencing data showed good quality and high reliability. Homology of the 48 868 unigenes were predicted by common databases NR (Non-Redundant), Swiss-port Protein Sequence Database, Eukaryotic Orthologous Groups of Proteins (KOG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and 25 797 unigenes were directly aligned onto the genes of apple (M. domestica). Compared with KOG database, the unigenes could be divided into 25 categories according to their function, including general function prediction for 19.45%, signal transduction mechanisms for 11.84%, and the cell motility was the least. Taking KEGG database as a reference, the unigenes were located to 145 branched metabolic pathways, including antibiotic biosynthesis for 842, purine metabolism for 740, starch and sucrose metabolism for 437, T cell receptors signaling pathways for 292. Then RSEM software was used to calculate the differential expression, and edgeR software was used for differential expression analysis. By setting the threshold false discovery rate (FDR) ＜0.05, log2(fold change (FC))＞ 2 or log2(FC)＜-2 to screen differentially expressed genes, all 1 230 upregulated genes and 1 869 downregulated genes were identified. The enrichment results of KEGG Pathway showed that 94 differential genes participated in tyrosine metabolism, plant-pathogen interaction, plant hormone signal transduction and biosynthesis of amino acids, etc.. Expression of some regulated genes validated by qRT-PCR showed consistence with transcriptome analysis. In this study, the second generation of high-throughput transcriptome sequencing technology was used to research the related genes of the Malus baccata in the resistence to Diplocarpon mali, which provides theoretical foundation for clearing the resistence mechanism.

Key words： Malus baccata Marssonina leaf blotch of apple Transcriptome High-throughput sequencing

收稿日期: 2016-08-19 出版日期: 2016-12-24

基金资助:马麝病毒性出血症的病原分离鉴定及其生物学特性研究;高等学校学科创新引智计划

通讯作者: 韩青梅 E-mail: hanqm@nwsuaf.edu.cn

引用本文:

李海录宋艳艳冯浩黄丽丽强晓玉韩青梅. 苹果褐斑病菌侵染苹果属山定子的转录组学分析[J]. , 2017, 25(1): 32-42.

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http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2017/V25/I1/32

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