拟轮枝镰孢与玉米籽粒互作的差异基因筛选及代谢通路分析

Screening of Differential Genes and Analysis of Metabolic Pathways in the Interaction Between Fusarium verticillioides and Maize Kernels

【目的】拟轮枝镰孢(Fusarium verticillioides)引起的玉米穗腐病是我国玉米产区发生严重的病害之一,论文旨在了解病原菌与玉米籽粒互作过程中的基因表达差异,为揭示病原菌的致病机制和玉米抗病机制提供依据。【方法】对拟轮枝镰孢侵染玉米籽粒0、4、12和72 h的样品进行转录组测序,之后采用生物信息学分析,分别以玉米和拟轮枝镰孢基因组为参考,以|log_(2)FC|≥1,P-adjust<0.05为阈值筛选互作过程中玉米和拟轮枝镰孢的差异表达基因,利用GO和KEGG对其进行功能注释及富集分析。Goatools软件分析植物-病原互作、MAPK途径和植物激素信号转导通路相关差异基因的表达变化,采用实时荧光定量PCR(qRT-PCR)方法对测序差异基因进行验证。【结果】在互作4、12和72 h后拟轮枝镰孢分别有140、400和1945个基因上调表达,有9、302和1784个基因下调表达;玉米分别有293、692和1426个基因上调表达,320、482和153个基因下调表达。GO和KEGG富集分析显示,侵染早期拟轮枝镰孢在细胞间隙生长,差异基因主要富集在RNA生物合成、细胞壁结构成分、脂肪酸生物合成、蛋白质代谢、碳水化合物代谢、生物过程和代谢过程等通路中。拟轮枝镰孢早期侵染触发了玉米活性氧(ROS)爆发,差异基因主要富集在对活性氧、过氧化氢的反应,几丁质酶、单加氧酶活性,木质素代谢过程等相关通路中。侵染后期拟轮枝镰孢继续在籽粒中定殖及扩展,差异基因主要富集在碳水化合物和细胞壁多糖分解代谢过程、跨膜转运、氧化还原酶活性等功能通路中。玉米主要通过苯丙素、木质素、类黄酮生物合成,MAPK信号通路、植物-病原互作和植物激素信号转导等途径差异基因大量表达响应病原菌侵染。随机选取6个玉米和6个拟轮枝镰孢的差异表达基因进行qRT-PCR分析,基因表达规律与转录组测序结果一致,证实了RNA-seq的准确性。【结论】在病原菌侵染早期,拟轮枝镰孢在细胞间隙生长,触发玉米活性氧爆发,相关通路差异基因表达;侵染中后期,病原菌以淀粉为营养素,继续在籽粒中定殖及扩展,玉米通过苯丙素、木质素及几丁质酶的生物合成等方面的相关基因表达响应拟轮枝镰孢侵染,同时植物-病原互作、MAPK途径和激素信号转导等途径参与抗拟轮枝镰孢侵染。

【Objective】Maize ear rot caused by Fusarium verticillioides is one of the most serious diseases in maize producing areas in China.The objective of this study is to understand the differences in gene expression during the plant-pathogen interaction at different stages,and to provide a basis for pathogenic mechanism of the pathogen infection and resistance mechanism of maize.【Method】Illumina platform was used to sequence the transcriptome of maize kernels infected with F.verticillioides at 0,4,12,and 72 h.The differentially expressed genes(DEGs)of maize and F.verticillioides were screened with|log_(2)FC|≥1,P-adjust<0.05 as threshold and clean reads were compared with genome of maize and F.verticillioides,separately.Functional annotation and enrichment analysis of DEGs were carried out by using GO and KEGG databases.Goatools software was used to analyze the expression changes of genes related to plant-pathogen interaction,MAPK signaling pathway and plant hormone signal transduction pathway.Sequencing results were verified by quantitative real-time PCR(qRT-PCR).【Result】A total of 140,400 and 1945 DEGs were up-regulated and 9,302,and 1784 DEGs were down-regulated in F.verticillioides after 4,12 and 72 h interaction,respectively.A total of 293,692,and 1426 DEGs were up-regulated and 320,482,and 153 DEGs were down-regulated in maize after 4,12 and 72 h interaction,respectively.GO and KEGG enrichment analysis of DEGs showed that F.verticillioides grew in intercellular space at the early stage of pathogen infection.The DEGs were enriched in RNA biosynthesis,cell wall structural component,fatty acid biosynthesis,protein metabolism,carbohydrate metabolism,biological process,and metabolic process.Reactive oxygen species(ROS)was triggered in maize at the early stage of infection.The DEGs were enriched in response to ROS,hydrogen peroxide,chitinase activity,monooxygenase activity,lignin metabolism.At the later stage of infection,F.verticillioides colonized and expanded in maize,and the DEGs were enriched in carbohydrate and cell wall polysaccharide catabolic process,transmembrane transport and oxidoreductase activity.Maize responded to pathogen infection through phenylpropanoid,lignin,flavonoid biosynthesis,MAPK signaling pathway,plant-pathogen interaction and plant hormone signal transduction.Six DEGs of maize and six DEGs of F.verticillioides were randomly selected for qRT-PCR.The results were consistent with those of transcriptome sequencing,which confirmed the accuracy of RNA-seq.【Conclusion】At the early stage of infection,F.verticillioides grew in the intercellular space,triggering ROS outbreak in maize and the expression of related pathway differential genes.At the middle and late stages of infection,the pathogen further colonized and expanded in maize with starch as nutrient.Maize responded to the infection of F.verticillioides through biosynthesis of phenylpropanoid,lignin and chitinase.Meanwhile,plant-pathogen interaction,MAPK signaling pathway,and plant hormone signal transduction were involved in the resistance to the infection of F.verticillioides.