丛枝菌根真菌响应桉树茄科雷尔氏菌侵染进程基因的筛选与鉴定
黄迪，毛健辉，王正木，张健朗，霍春宇，谢贤安，陈祖静
青枯病由茄科雷尔氏菌(Ralstonia solanacearum)引起，是世界上最具破坏性的细菌性病害之一。以化学防控、物理防控和改变栽培方式的方法防治青枯病的效果不理想。研究表明丛枝菌根真菌(AMF)能够防控植物病害，但其防控青枯病在多年生林木中的应用非常少。本研究以异形根孢囊霉(Rhizophagus irregularis)、茄科雷尔氏菌、巨桉(Eucalyptus grandis)三者之间不同的互作体系为研究对象，鉴定筛选菌根化巨桉幼苗根系中AMF响应青枯菌侵染进程关键基因信息。研究结果为研究AMF响应林木病原菌提供了一定的候选基因资源，也为利用AMF防控林木青枯病提供了一定的参考依据。
摘要
【背景】桉树(Eucalyptus)青枯病危害严重，丛枝菌根真菌(arbuscular mycorrhizal fungi, AMF)与桉树共生影响桉树对青枯病的抗性，而AMF响应桉树青枯菌侵染的机制仍不清楚。
【目的】探索AMF响应桉树茄科雷尔氏菌(Ralstonia solanacearum)的侵染机制。
【方法】以非菌根化和异形根孢囊霉(Rhizophagus irregularis)菌根化巨桉(Eucalyptus grandis)分别受茄科雷尔氏菌侵染0、24、48和96 h接种后(hour post-inoculated, hpi)的根系组织为研究对象，基于转录组测序筛选和鉴定菌根化巨桉根系中异形根孢囊霉响应茄科雷尔氏菌侵染的基因信息。
【结果】与对应非菌根化桉树受茄科雷尔氏菌侵染的时间点相比，菌根化桉树中异形根孢囊霉响应青枯菌侵染显著差异表达基因为3 382–5 989个，随青枯侵染时间进程的增加，异形根孢囊霉特异性响应茄科雷尔氏菌侵染差异表达基因数量逐渐增多。茄科雷尔氏菌侵染24 hpi时，异形根孢囊霉显著富集共生体生长、孢子形成和凋亡信号通路、铁载体等相关基因；茄科雷尔氏菌侵染48 hpi时，异形根孢囊霉主要提高自身跨膜运输的能力，促进自身钾、氮等养分吸收与交换等；茄科雷尔氏菌侵染96 hpi时，异形根孢囊霉主要调控氧化还原反应和黄酮类等抗菌物质合成。
【结论】菌根化桉树中AMF主要调控其生长加强生态位和营养(如氮、钾、铁)竞争、分泌抗菌物质、激活防御反应以响应青枯菌的侵染，所鉴定的相关基因信息为研究AMF-桉树-青枯菌互作机制提供一定的资源和参考。
图1 异形根孢囊霉在巨桉幼苗根系定殖 白色箭头为菌根共生结构. a：丛枝；v：泡囊. A：未接种异形根孢囊霉的巨桉根系. B：异形根孢囊霉在巨桉根系中形成丛枝. C：异形根孢囊霉在巨桉根系中形成泡囊
Figure 1 Rhizophagus irregularis colonized in roots of Eucalyptus grandis seedlings. The white arrow refers to the mycorrhizal symbiosis structure. a: Arbuscule; v: Vesicle. A: Root of E. grandis not inoculation with R. irregularis. B: R. irregularis formed arbuscular in the roots of E. grandis. C: R. irregularis fovesicles in the roots of E. grandis.
图2 巨桉根系AMF响应茄科雷尔氏菌侵染进程表达的基因数量 N_N：非菌根化巨桉幼苗侵染0 hpi；M_N：菌根化巨桉幼苗侵染0 hpi；M_R_24：菌根化巨桉幼苗受青枯菌侵染24 hpi；M_R_48：菌根化巨桉幼苗受青枯菌侵染48 hpi；M_R_96：菌根化巨桉幼苗受青枯菌侵染96 hpi
Figure 2 Number of genes expressed in AMF response to Ralstonia solanacearum infection in Eucalyptus grandis roots. N_N: Non-mycorrhizal E. grandis seedlings infection with R. solanacearum at 0 hpi; M_N: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 0 hpi; M_R_24: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 24 hpi; M_R_48: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 48 hpi; M_R_96: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 96 hpi.
图3 菌根化巨桉根系AMF响应茄科雷尔氏菌侵染24、48和96 hpi基因GO分析 M_N：菌根化巨桉幼苗；M_R_24：菌根化巨桉幼苗受青枯菌侵染24 hpi；M_R_48：菌根化巨桉幼苗受青枯菌侵染48 hpi；M_R_96：菌根化巨桉幼苗受青枯菌侵染96 hpi
Figure 3 GO analysis of AMF genes responded to the Ralstonia solanacearum infection at 24, 48 and 96 hpi in Eucalyptus grandis roots. M_N: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 0 hpi; M_R_24: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 24 hpi; M_R_48: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 48 hpi; M_R_96: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 96 hpi.
图4 菌根化巨桉幼苗中AMF响应茄科雷尔氏菌侵染24、48和96 hpi基因的KEGG分析 M_N：菌根化巨桉幼苗；M_R_24：菌根化巨桉幼苗受青枯菌侵染24 hpi；M_R_48：菌根化巨桉幼苗受青枯菌侵染48 hpi；M_R_96：菌根化巨桉幼苗受青枯菌侵染96 hpi
Figure 4 KEGG pathway of AMF responded to Ralstonia solanacearum infection at 24, 48 and 96 hpi in mycorrhizal Eucalyptus grandis seedlings. M_N: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 0 hpi; M_R_24: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 24 hpi; M_R_48: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 48 hpi; M_R_96: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 96 hpi.
图5 菌根化巨桉根系AMF响应茄科雷尔氏菌侵染进程抗性相关基因的表达 M_N：菌根化巨桉幼苗；M_R_24：菌根化巨桉幼苗受青枯菌侵染24 hpi；M_R_48：菌根化巨桉幼苗受青枯菌侵染48 hpi；M_R_96：菌根化巨桉幼苗受青枯菌侵染96 hpi
Figure 5 Expression of resistance related genes of AMF in mycorrhizal Eucalyptus grandis roots in responded to Ralstonia solanacearum infection process. M_N: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 0 hpi; M_R_24: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 24 hpi; M_R_48: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 48 hpi; M_R_96: Mycorrhizal E. grandis seedlings infection with R. solanacearum at 96 hpi.
设计制作：赵彬涵
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