中国农业科学 ›› 2017, Vol. 50 ›› Issue (17): 3323-3336.doi: 10.3864/j.issn.0578-1752.2017.17.007

• 植物保护 • 上一篇    下一篇

匍匐翦股颖接种立枯丝核菌后基因表达变化的转录组学分析

史毅,牛奎举,马晖玲   

  1. 甘肃农业大学草业学院/草业生态系统教育部重点实验室/中-美草地畜牧业可持续发展研究中心,兰州 730070
  • 收稿日期:2017-03-06 出版日期:2017-09-01 发布日期:2017-09-01
  • 通讯作者: 马晖玲,E-mail:mahl@gsau.edu.cn
  • 作者简介:史毅,E-mail:shiyi214@126.com
  • 基金资助:
    国家自然科学基金(31360583)

Transcriptome Analysis of Creeping Bentgrass (Agrostis stolonifera) Infected with Rhizoctonia solani

SHI Yi, NIU KuiJu, MA HuiLing   

  1. College of Pratacultural Science, Gansu Agricultural University/Key Laboratory of Grassland Ecosystem, Ministry of Education/Sino-U.S. Center for Grazingland Ecosystem Sustainability, Lanzhou 730070
  • Received:2017-03-06 Online:2017-09-01 Published:2017-09-01

摘要: 【目的】确定匍匐翦股颖(Agrostis stolonifera)接种立枯丝核菌(Rhizoctonia solani)后基因种类和表达量在转录水平的变化规律,明确草坪草病原菌侵染响应的关键基因。【方法】匍匐翦股颖生长14 d后采用麦粒培养物接种立枯丝核菌,接种3 d后选取感病叶片和未接种叶片提取RNA,进行转录组高通量测序,然后利用生物信息学分析,用Trinity组装匍匐翦股颖转录组,以组装子为参考,以|log2(fold change)|>1q-value<0.005为阈值选取感病和健康匍匐翦股颖叶片转录组的差异表达基因,并用iTAK软件分析其转录因子家族及表达变化,与植物R基因库进行blast分析R蛋白分类、利用Mapman软件分析生物胁迫信号通路相关基因的表达变化。【结果】高通量测序得到125 253 092条高质量待分析reads。经Trinity从头组装后,得到466 761条转录本。过半数的转录本长度为700 bp以上,组装结果N50=1 100 bp。使用CD-HIT选择334 212条转录本(所有转录本的71.60%)作为Unigene,平均长度573 bp,N50=791 bp。接种后植物比接种前植物基因有7 937个上调表达,1 570个下调表达。上调基因中296个,下调基因有142个都可被定义为转录因子,分布在58个转录因子家族中,其中锌指蛋白包含转录因子C2H2最多,有54个,C3H次之,为22个。差异基因中451个可定义为植物R蛋白表达基因,可分为33类,其中包含NBS-LRR结构域的抗病蛋白、LRR受体蛋白激酶、ABC-2类型的转运蛋白、U-box结构域蛋白激酶和热激蛋白这5类基因变化最显著。差异基因中大量上调表达基因可富集在病原识别、活性氧消除、信号传导、细胞凋亡、病程相关蛋白等生物胁迫相关基因类别,下调基因显著富集在植物生长发育相关类别和通路。qRT-PCR验证了随机挑选差异基因的表达量变化,均与RNA-seq分析结果一致,其中包括12个C2H2转录因子基因,10个C3H转录因子基因,以及12个R蛋白基因。【结论】病原菌侵染后,引起匍匐翦股颖大量基因表达变化,其中转录因子、R蛋白以及抗性相关基因多为上调表达,作用为抑制病原菌扩散,而生长发育相关基因表达下调,这些进程共同使匍匐翦股颖产生对立枯丝核菌的先天基础抗性。

关键词: 匍匐翦股颖, 立枯丝核菌, 转录组, 差异表达基因, 抗病机制

Abstract: 【Objective】The objective of this study is to reveal the gene expression pattern of creeping bentgrass (Agrostis stolonifera) after been inoculated with Rhizoctonia solani, by comparing the gene expression level between plant with disease and without disease during gene transcription, and to identify the key genes of turfgrass responding to pathogen infection. 【Method】A. stolonifera which was grown for 14 days, was inoculated with R. solani. Leaf samples of plant with or without disease were collected after 3 days. High-throughput sequencing technology and Trinity software were used for obtaining the transcriptome. Different expression genes were screened with |log2 (fold change)| >1, q-value<0.005 as threshold and the assembly was used as reference. Bioinformatics software was used to analyze the transcriptome difference. iTAK was used for transcription factors. All the DEGs were blasted with plant resistance gene database to find the R protein. Mapman software was used to analyze the signaling pathway. 【Result】 Using the high-throughput transcriptome sequencing, 125 253 092 were obtained. 466 761 transcripts were assembled by Trinity software. More than half of them were longer than 700 bp and N50=1 100 bp. CD-HIT was used to choose 334 212 transcripts as Unigene with the average length of 573 bp and N50=791 bp. By comparing the A. stolonifera transcripts with disease and without disease, 7 937 up-regulated genes and 1 570 down-regulated genes were obtained. Among the up-regulated genes, 296 of them are transcription factors (TFs), while 142 of down-regulated genes can be defined as TFs. Those TFs were classified as 58 families, including 54 zinc-finger protein containing TFs C2H2, which was the most, then 22 C3H TFs. A total of 451 of different expression genes (DEGs) can be annotated as plant R-protein, which can be classified into 33 families. Among these families, NBS-LRR containing resistance protein, LRR-like receptor protein kinase, ABC-2 type transporter, U-box domain containing protein kinase, and heat shock protein gene, these 5 families showed the most variation. A bunch of up-regulated DEGs can be mapped in plant biotic response pathway and enriched in pathogen recognition, ROS eliminate, signaling transport, programmed cell death, pathogenesis-related protein and so on, while the down-regulated genes were enriched in plant growth and development pathways. The expression level of random selected DEGs was tested with qRT-PCR, and they were consistent with the result of RNA-seq analysis. The random selected genes included 12 C2H2 TFs, 10 C3H TFs and 12 R-protein genes.【Conclusion】The inoculation of pathogen caused great expression difference of A. stolonifera transcriptome. Most of the TFs, R-protein and defense-related genes were up-regulated to suppress pathogen growth. The inherent resistance of A. stolonifera to R. solani has been generated with the co-action of all these genes.

Key words: Agrostis stolonifera, Rhizoctonia solani, transcriptome, different expression genes, disease resistance mechanism