| 摘要: |
| 【目的】探究核盘菌响应环磷酸腺苷（cyclic adenosine monophosphate，cAMP）处理的转录组学特征，挖掘调控核盘菌菌核形成的关键基因，为靶向杀菌剂的研发提供参考。【方法】以接种在PDA培养基上生长至菌核原基形成时期的核盘菌作为CK1，开始形成黑色素时期的核盘菌作为CK2，以接种在PDA+5 mmol/L cAMP培养基上生长相同时间(5,7 d)的核盘菌作为试验组，依次命名为M1 和M2，对这4组样品进行转录组测序（RNA-seq）比较，阐释cAMP影响菌核形成的调控途径，挖掘菌核原基及菌核黑色素形成中的差异表达基因（differentially expressed genes，DEGs）。【结果】外源添加的cAMP主要在M1前期影响胞内的cAMP含量。差异表达基因分析结果显示，M1和CK1之间共有681个差异表达基因；KEGG富集分析显示，这些基因主要参与各类氨基酸代谢、脂肪酸代谢、MAPK信号通路等，且通路中的大部分基因都呈上调表达模式，而MAPK及其下游作用因子钙调磷酸酶B亚基（calcineurin B subunit，CnB）和热休克蛋白70（heat shock 70 ku protein，HSP70）则明显下调。M2和CK2之间共筛选到4 490个差异表达基因，主要参与各类氨基酸代谢、抗原加工提呈以及cAMP、MAPK等信号通路和自噬途径，通路中的大部分基因呈上调表达模式，且自噬相关因子ATG2、ATG20、ATG22、ATG23、ATG27和ATG29也大量上调表达，而与蛋白质和脂类合成相关的磷酸酶B（phosphatase B，PTPB）和酰基辅酶A氧化酶（acyl coenzyme A oxidase，ACO）的表达量则明显下调。在M1和M2时期，相关基因的qRT-PCR与RNA-seq差异表达分析结果趋势相同，说明转录组测序数据准确。【结论】核盘菌可通过吸收胞外高浓度的cAMP抑制菌核形成菌丝，其抑制作用主要通过影响相关蛋白质及脂类物质的合成而对菌丝形态及信号传递造成障碍。 |
| 关键词: 核盘菌 菌核形成 环磷酸腺苷 差异表达基因 |
| DOI： |
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| 基金项目:陕西省自然科学基础研究计划项目(2021JQ-725)；陕西中医药大学学科创新团队项目（2019-QN01） |
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| Analysis of differentially expressed genes in sclerotial formation of Sclerotinia sclerotiorum inhibited by cAMP |
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LÜ Ruihua,FENG Zhao,LÜ Ruihua，et al
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| Abstract: |
| 【Objective】This study explored transcriptomic characteristics of Sclerotinia sclerotiorum in response to cyclic adenosine monophosphate (cAMP) treatment and excavated key genes regulating sclerotia formation to provide reference for the development of targeted fungicides.【Method】This study used S. sclerotiorum on PDA medium until sclerotia primordia formation as CK1,S. sclerotiorum starting to form melanin as CK2,and S. sclerotiorum on PDA+5 mmol/L cAMP medium for same days (5 and 7 days) as the experimental groups of M1 and M2.Transcriptome sequencing (RNA seq) was performed on samples from these four groups to elucidate regulatory pathways of cAMP affecting sclerotial formation and dig for differentially expressed genes (DEGs) in the formation of sclerotia primordia and melanin.【Result】Exogenous cAMP mainly affected intracellular cAMP contents in the early stage of M1.M1 and CK1 obtained a total of 681 DEGs.KEGG enrichment analysis showed that these genes mainly involved in various amino acid metabolism,fatty acid metabolism and MAPK signaling pathways.Most genes in these pathways showed an upregulated expression pattern,while MAPK and its downstream effector factors of calcineurin B subunit (CnB) and heat shock protein 70 (HSP70) were significantly downregulated.The 4 490 DEGs screened by M2 and CK2 mainly involved in various amino acid metabolism,antigen processing and presentation,signaling pathways of cAMP and MAPK,as well as autophagy pathways.Most genes exhibited an upregulation pattern,and autophagy related factors of ATG2,ATG20,ATG22,ATG23,ATG27 and ATG29 were also upregulated in large quantities.Expression levels of phosphatase B (PTPB) and acyl coenzyme A oxidase (ACO) related to protein and lipid synthesis were significantly downregulated.The qRT-PCR and RNA-seq analysis of related factors at M1 and M2 stages showed same trends,indicating that the transcriptome sequencing data was accurate.【Conclusion】The mycelia of S. sclerotiorum inhibited sclerotia formation by absorbing high concentrations of cAMP outside cells. The inhibitory effect was mainly caused by affecting the synthesis of related proteins and lipids and hindering the morphology and signal transmission of mycelium. |
| Key words: Sclerotinia sclerotiorum sclerotia formation cyclic adenosine monophosphate (cAMP) differentially expressed genes |
