【作者】 张丹；

【导师】 方卫国；

【作者基本信息】 浙江大学 ， 微生物学， 2021， 硕士

【摘要】 昆虫病原真菌对宿主的入侵是一个非常复杂的过程,正常情况下,宿主微生物群在宿主体内维持着动态的生态平衡。一旦昆虫病原真菌入侵,宿主内微生态平衡会被打破,宿主-病原真菌-宿主微生物群落三者间会发生复杂的相互作用。现阶段,昆虫病原真菌与宿主之间的互作已进行了大量研究,但对昆虫病原真菌与宿主微生物群落互作的认识还非常有限。发现并阐明昆虫病原真菌与宿主微生物群落之间的互作模式对全面认识昆虫病原真菌的致病机制具有重要意义,可为开发绿色高效稳定的真菌杀虫剂提供理论依据。罗伯茨绿僵菌（Metarhizium robertsii）的寄主范围非常广泛,可成功感染几百种不同种类的昆虫,是研究昆虫病原真菌与宿主微生物群落互作的良好材料。实验室前期的研究发现,罗伯茨绿僵菌通过水平基因转移（horizontal gene transfer,HGT）获得包括MrVoc1在内的18个基因,但MrVoc1基因的具体功能还未被研究清楚。本研究发现MrVoc1参与罗伯茨绿僵菌与昆虫微生物群落的互作过程。主要研究结果如下:1)前期研究发现,MrVoc1对罗伯茨绿僵菌的杀虫毒力无贡献。但q RT-PCR显示MrVoc1在大蜡螟血淋巴条件下高水平表达。本研究分析罗伯茨绿僵菌侵染致死的大蜡螟尸体发现,大蜡螟被罗伯茨绿僵菌侵染致死后呈红色的僵虫状;保湿10～15d后,根据表面微生物的生长情况可将大蜡螟尸体分为以下四类。类型Ⅰ:尸体是僵虫,其表面几乎全部被罗伯茨绿僵菌的孢子覆盖。类型Ⅱ:尸体是僵虫,其表面被菌丝覆盖,只有少量的罗伯茨绿僵菌孢子。类型Ⅲ:虫尸不僵硬或部分僵硬,其上除了有罗伯茨绿僵菌孢子和菌丝之外,还有其它真菌或细菌生长,并有黑色液体流出。类型Ⅳ:尸体是僵虫,其上只有较少的罗伯茨绿僵菌菌丝体。WT感染致死的虫尸中,四种类型虫尸的比例分别为46%、29%、22%和3%;ΔMrVoc1感染致死的虫尸中,四种类型虫尸的比例分别为26%、38%、32%和4%。ΔMrVoc1感染的类型Ⅰ虫尸的比例显著低于WT;ΔMrVoc1感染的类型Ⅱ虫尸的比例显著高于WT;ΔMrVoc1感染的类型Ⅲ虫尸的比例显著高于WT。2)从罗伯茨绿僵菌感染的昆虫体内分离出两株细菌——GMG1和GMG2,通过16S r RNA测序和系统发育树分析进行鉴定,推测这两株细菌分别为Staphylococcus属和Proteus属的一员。WT感染致死的大蜡螟尸体提取物对GMG1和GMG2的抑制作用均显著大于ΔMrVoc1。通过硅胶层析色谱分离大蜡螟尸体提取物,发现MrVoc1的敲除使感染的大蜡螟尸体提取物中能够抑制GMG1或GMG2的部分抑菌物质消失。3)MrVoc1不影响罗伯茨绿僵菌和大肠杆菌对抗生素的抗性。在博来霉素存在的条件下,ΔMrVoc1菌株、MrVoc1^OE菌株以及C-ΔMrVoc1菌株的生长速率与WT菌株无显著差异。原核表达MrVoc1蛋白不影响大肠杆菌BL21菌株对博来霉素、链霉素、妥布霉素、庆大霉素、青霉素、氨苄青霉素和头孢噻肟的抗性。在利福平存在的条件下,无论是否添加IPTG,BL21菌株、转入pET-28a空载或pET-28a-MrVoc1的BL21菌株均能生长,但转入pET-28a空载或pET-28a-MrVoc1的BL21菌株在添加IPTG的条件下,生长都受到一定程度的抑制,且转入pET-28a-MrVoc1的BL21菌株的生长抑制效果更显著;在潮霉素B存在的条件下,无论是否添加IPTG,BL21菌株均能生长,转入pET-28a空载或pET-28a-MrVoc1的BL21菌株在不添加IPTG的条件下均能生长,但转入pET-28a空载或pET-28a-MrVoc1的BL21菌株在添加IPTG的条件下,生长均受到抑制。纯化的MrVoc1蛋白与潮霉素B的反应产物对BL21无抑制作用;纯化的MrVoc1蛋白与利福平的反应产物对BL21也无抑制作用。4)MrVoc1的过表达使罗伯茨绿僵菌的代谢物组发生改变,WT菌株中含有甲基乙二醛（MG）等代谢物,MrVoc1^OE菌株中不存在。罗伯茨绿僵菌入侵后,大蜡螟体内MG含量显著升高,且WT菌株和MrVoc1^OE菌株侵染致死的大蜡螟尸体中MG含量显著高于ΔMrVoc1菌株。MG能抑制罗伯茨绿僵菌及GMG1和GMG2的生长,MG对GMG1和GMG2的抑制作用比对罗伯茨绿僵菌更显著。此外,喂食MG的白纹伊蚊的肠道菌群总数量显著高于正常的白纹伊蚊,且两组白纹伊蚊肠道中的优势菌群也显著不同。综上,本研究发现MrVoc1参与罗伯茨绿僵菌与昆虫微生物群落的互作过程,进一步挖掘了昆虫病原真菌与宿主微生物群落之间互作的部分机制,加深了对病原真菌-宿主微生物群落-宿主三者间互作的认识。更多还原

【Abstract】 The invasion of entomopathogenic fungi into the host is a very complex process.Under normal circumstances,the host microbiota maintains a dynamic ecological balance.Once the entomopathogenic fungi invade,host microecological balance will be broken and the complex interaction between the host,pathogenic fungi and host microbiota will occur.At present,the interaction between entomopathogenic fungi and their hosts has been studied extensively,but little is known about the interaction between entomopathogenic fungi and host microbiota.Moreover,discovering and elucidating the interaction mechanism between entomopathogenic fungi and host microbiota is of great significance for comprehensively understanding the pathogenic mechanism of entomopathogenic fungi,which can provide theoretical basis for the development of co-friendly,efficient and stable fungal insecticides.The broad host-range entomopathogen Metarhizium robertsii can infect hundreds of insect species,hence it’s a good material for studying the interaction between entomopathogenic fungi and the host microbiota.Previous studies showed that M.robertsii obtained 18 genes including MrVoc1 via horizontal gene transfer（HGT）,but the function of MrVoc1 gene has not been clearly studied.In this study,we found that MrVoc1 was involved in the interaction between M.robertsii and insect microbiota.The main research results are as follows:1)Previous studies found that MrVoc1 did not contribute to the virulence of M.robertsii.However,q RT-PCR result showed that MrVoc1 was highly expressed during colonizing of the hemolymph of Galleria mellonella.In this study,we observed G.mellonella cadavers,and found that cadavers mummified by M.robertsii were red and stiff.According to the growth of microbiota on the surface,G.mellonella cadavers moisturized 10～15 days can be divided into the following four types.TypeⅠ:the cadavers were stiff and almost entirely covered with M.robertsii spores.TypeⅡ:the cadavers were stiff and covered with mycelia of M.robertsii.TypeⅢ:the cadavers were not stiff or partially stiff.In addition to the spores and hyphae of M.robertsii,there were other fungi or bacteria growing and black fluid flowing out.TypeⅣ:the cadavers were stiff and covered with few mycelia of M.robertsii.Among G.mellonella cadavers infected by WT,typeⅠ、Ⅱ、ⅢandⅣrespectively accounted for 46%、29%、22%and 3%.Among G.mellonella cadavers infected byΔMrVoc1,typeⅠ、Ⅱ、ⅢandⅣrespectively accounted for 26%、38%、32%and 4%.The proportion of typeⅠcadavers mummified byΔMrVoc1 was significantly lower than WT;the proportion of typeⅡcadavers mummified byΔMrVoc1 was significantly higher than WT;the proportion of typeⅢcadavers infected byΔMrVoc1 was significantly higher than WT.2)GMG1 and GMG2 were isolated from the insects infected by M.robertsii,and were identified by 16S r RNA sequencing and phylogenetic tree analysis.We speculated that the two strains were members of Staphylococcus and Proteus,respectively.Besides,we recognized the antimicrobial activity of the extracts from G.mellonella cadavers infected by WT was significantly higher than that ofΔMrVoc1.Disruption of MrVoc1 resulted in the disappearance of some antimicrobial substances of extracts from G.mellonella cadavers that could inhibit GMG1 or GMG2.3)MrVoc1 had no effect on antibiotic resistance of M.robertsii and E.coli.On PDA plates with bleomycin,the growth rates ofΔMrVoc1,MrVoc1^OE and C-MrVoc1were not significantly different from WT.Prokaryotic expression of MrVoc1 protein had no effect on the antimicrobial activity of bleomycin,streptomycin,tobramycin,gentamicin,penicillin,ampicillin and cefotaxime against BL21.In the presence of rifampicin,BL21 and strains with plasmids pET-28a or pET-28a-MrVoc1 could grow with or without IPTG.However,the growth of BL21 with plasmids pET-28a or pET-28a-MrVoc1 with IPTG was inhibited to a certain extent,and the antibacterial activity against BL21 with plasmids pET-28a-MrVoc1 was more stronger.In the presence of hygromycin B,BL21 could grow with or without IPTG,and BL21 with plasmids pET-28a or pET-28a-MrVoc1 could grow without IPTG.However,the growth of BL21 with plasmids pET-28a or pET-28a-MrVoc1 in the presence of IPTG was inhibited.The MrVoc1 protein purified was co-incubated with hypomycin B/rifampicin at 28℃,and we found that hypomycin B/rifampicin and the reaction products of hypomycin B/rifampicin-MrVoc1 protein did not inhibit the growth of BL21.4)Overexpression of MrVoc1 altered the metabolome of M.robertsii.Metabolites such as methylglyoxal were found in WT,but not in MrVoc1^OE.The MG content in G.mellonella cadavers mummified by WT was significantly increased.Moreover,the MG content in G.mellonella cadavers mummified by WT and MrVoc1^OEwas significantly higher than that ofΔMrVoc1.MG inhibited both the growth of M.robertsii,GMG1 and GMG2,but the antimicrobial activity of MG against GMG1 and GMG2 was stronger than that of MG against M.robertsii.In addition,the number of the intestinal flora of Aedes albopictus fed with MG was significantly higher than that of normal A.albopictus,and the dominant microflora of the two groups was also significantly different.In conclusion,we found that MrVoc1 mediated the interaction between M.robertsii and insect microbiota,and we further elucidated part of the mechanism of the interaction between entomopathogenic fungi and host microbiota.This deepens our understanding of the interactions between host,pathogenic fungi and host microbiota.更多还原