转录组分析罗伯茨绿僵菌精胺合成酶MrSPS介导真菌血腔定殖功能

Transcriptomic analysis of Metarhizium robertsii during hemocoel colonization after deletion of a spermine synthase MrSPS

摘要:

【背景】精胺在植物应对逆境胁迫、动物抵抗疲劳和衰老、真菌生长代谢等过程中发挥重要作用，但目前在昆虫病原真菌中的研究未见报道。【目的】在分子水平上探究罗伯茨绿僵菌精胺合成关键酶——精胺合成酶在昆虫血腔定殖中的作用机制。【方法】显微注射法测定Mrsps敲除株ΔMrsps的致病力变化，并观察血腔中ΔMrsps生长状态；收集ΔMrsps和野生型WT注射侵染30 h后的大蜡螟血淋巴进行转录组测序，分别与罗伯茨绿僵菌和大蜡螟参考基因组进行比对分析，并结合定量PCR进行验证。【结果】与WT和回补株ΔMrsps-cp相比较，ΔMrsps致病力显著下降，而且随着注射浓度的降低，ΔMrsps致病力下降越显著。侵染36 h后WT和ΔMrsps孢子都能正常萌发且开始以类酵母状态生长，60 h后，相较于WT，ΔMrsps的生长繁殖数量较少。转录组共检测到3 202个罗伯茨绿僵菌基因，其中1 769个基因在ΔMrsps中表达上调，922个基因表达下调；差异表达基因涉及碳水化合物代谢、运输、分解代谢、翻译和氨基酸代谢等多条途径；筛选出28个血腔致病相关基因全部在ΔMrsps中表达下调；定量PCR检测发现在整个血腔定殖阶段免疫逃避蛋白Mcl1基因和血腔定殖Colonization of hemocoel 1基因在WT和ΔMrsps-cp中的表达量高于ΔMrsps。共检测到13 249个大蜡螟基因，其中4 026个差异表达基因；KEGG注释分析显示大量差异表达基因富集到内分泌系统和免疫系统等途径；深入分析发现22个差异表达基因归属于Toll和Imd信号通路，其中18个基因在ΔMrsps侵染的大蜡螟中表达上调，表明ΔMrsps侵染大蜡螟过程中更易引起免疫系统的激活。【结论】揭示了Mrsps在罗伯茨绿僵菌血腔定殖阶段作用的分子机制，为进一步揭示精胺在真菌中的作用机理提供了理论基础。

Abstract:

[Background] Spermine plays an important role in plant defense response, animal anti-fatigue and anti-aging fight, and fungal growth and metabolism. However, there have been few reports on spermine function in the entomopathogenic fungi. [Objective] The study explored the molecular mechanism of spermine synthase (MrSPS) from Metarhizium robertsii during insect hemocoel colonization. [Methods] Insect bioassays of Galleria mellonella larvae were performed to examine the virulence of Mrsps deleted M. robertsii (ΔMrsps), and the growth characteristics of ΔMrsps in hemolymph were observed. Hemolymph of G. mellonella larvae injected with ΔMrsps and wild-type (WT) strains for 30 h were collected for transcriptome sequencing, and comparisons were made with genes from M. robertsii and G. mellonella separately. Real-time quantitative PCR was performed for verification of transcriptome sequencing. [Results] Compared to WT and complementation (ΔMrsps-cp) strains, ΔMrsps showed significantly decreased virulence, and the pathogenicity of ΔMrsps reduced markedly with decreasing of the injection concentration. After 36 h of infection, spores from both WT and ΔMrsps germinated normally and began to grow in a yeast-like state, while ΔMrsps biomass was less than that of WT after 60 h. A total of 3 202 genes were detected from M. robertsii, of which 1 769 were up-regulated and 922 were down-regulated in ΔMrsps. Lots of differentially expressed genes (DEGs) were involved in carbohydrate metabolism, and transport, catabolism, translation, and amino acid metabolism pathways. All identified 28 genes related to hemocoel colonization from DEGs were down-regulated in ΔMrsps. Real-time quantitative PCR showed that both collagen-like protein and Colonization of hemocoel 1 genes were decreased during the whole stage of hemocoel colonization in ΔMrsps compared to the conditions in WT and ΔMrsps-cp. A total of 13 249 genes were detected from G. mellonella, of which 4 026 genes had differential expression. KEGG analysis revealed most of DEGs were enriched in endocrine system and immune system. Furthermore, among the 22 genes involved in Toll and Imd signaling pathways, 18 were up-regulated in G. mellonella infected by ΔMrsps, which suggested ΔMrsps was more likely to cause the activation of the insect immune system than WT. [Conclusion] The molecular mechanism of MrSPS function in hemocoel colonization of M. robertsii was explored for the first time, which provided a theoretical basis for further revealing the mechanism of spermine function in fungi.