虫尸表面罗伯茨绿僵菌产孢、致病力及其相关基因的表达特征

Sporulation, pathogenicity and transcriptomic analysis of entomopathogenic fungus Metarhizium robertsii growing on the surface of mycosed insect cadavers

中文关键词:绿僵菌  虫尸表面  产孢  致病力  丝裂原活化蛋白激酶  级联信号通路

keywords:Metarhizium  insect cadaver surface  sporulation  pathogenicity  MAPK signaling pathway

中文摘要:为明确绿僵菌在其致死害虫表面生长发育和致病力等相关分子机制，通过显微观察大蜡螟Galleria mellonella虫尸表面罗伯茨绿僵菌Metarhizium robertsii生长和产孢特性，并测定其致病力，分别对虫尸表面和PDA培养基上罗伯茨绿僵菌的菌丝生长阶段和大量产孢阶段进行高通量测序，对虫尸表面罗伯茨绿僵菌产孢及致病力通路相关基因进行系统分析，并采用荧光定量PCR技术对高通量测序结果进行验证。结果表明，PDA培养基上培养5 d后，罗伯茨绿僵菌开始大量产孢，培养14 d时产孢量最高，为4.6×10⁷个/cm²，大蜡螟幼虫注射罗伯茨绿僵菌4 d后，其体表出现菌丝，5.5 d后虫尸表面罗伯茨绿僵菌大量产孢，9 d后产孢量最高，为2.6×10⁸个/cm²。与PDA培养基上罗伯茨绿僵菌对大蜡螟幼虫的半致死时间（7.09 d和4.66 d）相比，体壁侵染法和显微注射法侵染的虫尸表面罗伯茨绿僵菌对大蜡螟幼虫的半致死时间（6.33 d和4.49 d）分别显著缩短和无显著变化。高通量测序结果显示，在菌丝生长阶段和大量产孢时期，虫尸表面罗伯茨绿僵菌中共有810个基因上调表达，452个基因下调表达，其中大量差异基因富集到丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）级联信号、ABC转运蛋白、类固醇生物合成及甘油磷脂代谢等通路中。在虫尸表面罗伯茨绿僵菌MAPK级联信号通路中，在菌丝生长和大量产孢阶段分别有36个和32个基因发生显著差异表达。在MAPK调控通路中，虫尸表面罗伯茨绿僵菌在菌丝生长和大量产孢阶段分别有11个和12个产孢相关基因，有11个和8个致病力相关基因发生差异表达，荧光定量PCR结果与高通量测序结果高度一致。

Abstract:In order to explore the growth, development and pathogenicity mechanism of entomopathogenic fungus Metarhizium robertsii growing on the surface of dead insects, the growth, sporulation characteristics and pathogenicity of the fungus growing on the surface of mycosed Galleria mellonella cadavers were analyzed with microscopic observation and virulence assays. The fungi during hyphal growth and mass sporulation were collected from the surface of the mycosed insect cadavers and PDA medium for high-throughput sequencing. Moreover, a systematic analysis of the genes involved in sporulation and pathogenicity was performed by high-throughput sequencing and quantitative RT-PCR. The results showed that a large number of spores were produced at 5 d, and maximum sporulation was observed in M. robertsii grown on PDA medium on day 14 (4.6×10⁷ spores/cm²). Hyphae appeared on the surface of insects at 4 d after injection with the fungal spores; a large number of spores were produced at 5.5 d, and maximum sporulation was observed on day 14 (2.6×10⁸ spores/cm²). The median lethal time of M. robertsii from the surface of the mycosed insect cadavers (6.33 d) was shorter than that on PDA medium (7.09 d) by topical inoculation, but there was no significant difference between them (4.66 d and 4.49 d) by spore injection assays. High-throughput sequencing showed that 810 up-regulated and 452 down-regulated genes were detected in M. robertsii grown on the mycosed insect cadavers. A plenty of genes were involved in mitogen-activated protein kinase (MAPK) signaling pathways, ABC transporters, steroid biosynthesis and glycerophospholipid metabolism pathways. Further analysis showed that 36 and 32 differential expressed genes involved in MAPK signaling pathways were identified between M. robertsii collected from the surface of mycosed insect cadavers and PDA medium during hyphal growth and mass sporulation stages. Moreover, compared with the genes of M. robertsii collected from PDA medium, 11 and 12 genes involved in sporulation, and 11 and eight genes involved in pathogenicity were differentially expressed in M. robertsii collected from the surface of mycosed insect cadavers during hyphal growth and mass sporulation stages. The expression profiles of the differentially expressed genes involved in sporulation and pathogenicity were highly consistent with the sequencing data.