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

发布时间：2018-02-23 02:53

本文关键词： 匍匐翦股颖立枯丝核菌转录组差异表达基因抗病机制　出处：《中国农业科学》2017年17期 　论文类型：期刊论文

【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)|1,q-value0.005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i TAK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。q RT-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。
[Abstract]:[objective] to determine the variation of gene types and expression levels at transcription level after inoculation of Agrostis stolonifera with Rhizoctonia solaniae (Rhizoctonia solani). [methods] after 14 days of growth of creeping bentgrass, Rhizoctonia solani was inoculated with wheat grain culture, and RNA was extracted from susceptible and uninoculated leaves 3 days later, and high throughput sequencing was carried out. Then using bioinformatics analysis, Trinity was used to assemble the transcriptome of bentgrass, with the assembler as reference, and log2(fold exchange) 1nq-value0.005 as the threshold to select the differentially expressed genes of leaf transcriptome of susceptible and healthy creeping bentgrass. I TAK software was used to analyze the transcription factor family and its expression changes, and blast was used to analyze the classification of R protein with the plant R gene bank. Mapman software was used to analyze the expression changes of genes associated with biological stress signaling pathway. [results] 125,253,092 high-quality READswere obtained by high-throughput sequencing. 466,761 transcripts were obtained. More than half of the transcripts were more than 700 BP in length. CD-HIT was used to select 334,212 transcripts (71.60 of all transcripts) as Unigenes.The average length of CD-HIT was 573bpN ~ (50,791) bp.There were 7,937 up-regulated genes and 1,570 down-regulated genes after inoculation, and 296 of them were up-regulated genes. 142 down-regulated genes can be defined as transcription factors, distributed in 58 transcription factor families, among which zinc finger protein contains the transcription factor C2H2, followed by 54 C3H. There are 451 genes that can be defined as plant R protein expression genes, which can be classified into 33 classes. The most significant changes were found in the five types of genes including NBS-LRR domain resistance protein kinase, ABC-2 type transporter, U-box domain protein kinase and heat shock protein. A large number of up-regulated genes in differentially expressed genes can be enriched in pathogen recognition. Reactive oxygen species (Ros) elimination, signal transduction, apoptosis, pathogenesis-related proteins, and down-regulated genes were significantly enriched in plant growth and development related classes and pathways. Q RT-PCR confirmed the changes in the expression levels of randomly selected differentially expressed genes. All the results were consistent with the results of RNA-seq analysis, including 12 C2H2 transcription factor genes, 10 C3H transcription factor genes and 12 R protein genes. [conclusion] after infection by pathogenic bacteria, a large number of genes expressed in creeping bentgrass were changed. The transcription factor R protein and resistance-related genes were upregulated and inhibited the spread of pathogen, but the expression of growth-development related genes was down-regulated. These processes led to the innate basic resistance against Rhizoctonia solani in creeping bentgrass.
【作者单位】：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;
【基金】：国家自然科学基金(31360583)
【分类号】：S436.8

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