【作者】 刘静；

【导师】 万永继；

【作者基本信息】 西南大学 ， 微生物学， 2021， 博士

【摘要】 球孢白僵菌是一种重要的昆虫病原真菌,对昆虫的致病过程大约可以分为三个阶段,即表皮穿透、在昆虫血液中定殖和生长、再次穿透宿主体表菌丝由体内生长至体外进行生殖繁育。迄今在球孢白僵菌各侵染阶段已鉴定出大量的相关基因,主要目标集中于提高真菌的毒力和生物防治。在侵染过程中,球孢白僵菌可以通过昆虫表皮感染宿主,且宿主广泛,但分离于特定宿主的球孢白僵菌在感染不同宿主昆虫时表现出致病性的显著差异,造成致病性差异的分子机理及如何适应寄生不同昆虫宿主环境的分子机制仍不清楚,寻找与发现球孢白僵菌在侵染不同宿主过程中相关寄生适应性功能的基因是本研究的主要目的。通过比较转录组学的方法分析了球孢白僵菌响应不同昆虫宿主的基因表达特征,本研究揭示了球孢白僵菌的特定菌株对不同昆虫宿主的致病性差异的分子机理,同时发现了球孢白僵菌适应不同宿主的一些特殊差异表达基因,如过敏原（应变原,Bb-f2,BBA＿05395）基因和III型几丁质酶Class III chitinase Chi A2基因（Bbchi-17,BBA＿05353）等,特别是分离于家蚕的高毒力菌株（GXsk1011）在响应其它昆虫宿主的表皮时其过敏原蛋白基因Bb-f2为显著上调表达,通过基因敲除和超量表达的方法,研究了Bb-f2基因的生物学功能和效应,并进一步通过异源真核细胞表达Bb-f2并将该蛋白注射到家蚕体内,检测了对家蚕Toll通路中模式识别蛋白、受体蛋白、胞质信号转导因子及抗菌肽基因的表达特征,分析和探索了Bb-f2蛋白在球孢白僵菌侵染昆虫过程中的作用机制。主要研究结果如下:1、球孢白僵菌GXsk1011对家蚕、棉铃虫和豆青虫的致病性以球孢白僵菌GXsk1011侵染3种鳞翅目昆虫,五龄家蚕、棉铃虫和豆青虫,在接种浓度为1×10⁸ conidia/m L条件下,球孢白僵菌对家蚕、棉铃虫和豆青虫的感染死亡率分别为100%、52.50%和8.88%;测定家蚕、棉铃虫和豆青虫的LC₅₀依次为1.25×10⁶,4.67×10⁷和5.25×10¹⁰conidia/m L。结果表明球孢白僵菌对原始分离（易感）宿主家蚕和两种非原始分离宿主的毒力具有显著性差异。2、球孢白僵菌对3种鳞翅目昆虫表皮响应的差异表达基因为了揭示致病性差异的原因,对球孢白僵菌响应3种昆虫表皮的转录组学数据进行了分析。由于早期感染阶段（24 h）菌体生物量太低,以及为了避免宿主活体组织RNA酶的消化作用及其它微生物的影响,采用了体外模拟感染试验的方案及RAN-seq,其转录组数据由本研究室登录在NCBI数据库（登录号:SRR3036777,SRR3043073和SRR3043097）。本研究对测序数据进行了基因注释,对转录组测序数据分析表明:两两比较上调表达的差异基因数量约为600-900个,下调基因的数量约为400-800个。通过对差异基因的注释,其差异基因主要涉及到菌株的代谢、催化活性、转运子活性、定位等生物学过程和通路,鉴定获得了一些与致病性差异密切相关的关键毒力基因以及菌株对不同宿主表皮响应的基因表达模式。对差异最高的前20个差异表达基因（Top 20）的分析表明:在球孢白僵菌响应敏感宿主家蚕时,涉及感染过程的关键因子即对体壁的黏附、表皮蛋白降解以及一些生长类基因为显著上调表达,如:黏附蛋白（Mad 1）、类胰蛋白酶（Pr2）,氧甲基转移酶（O-methyltrans-ferase,family 3,BBA＿09875）及细胞表面蛋白（cell surface protein,BBA＿09174）等,且在Top 20基因之外,对表皮有降解作用的Endothiapepsin-like天冬氨酸蛋白酶基因（Bbenp L-1）等也是上调表达,由于这些毒力基因的高量表达使其菌株对家蚕表现为高的感染致病性（致死率100%）;但当球孢白僵菌对非原始宿主棉铃虫和豆青虫响应时,显著上调的基因是过敏原蛋白（Bb-f2,BBA＿05395,major allergen Asp f 2-like protein）、III型几丁质酶（Bbchi-17,BBA＿05353,class III chitinase Chi A2）、非核糖体肽合成酶（nonribosomal peptide synthase,BBA＿08424）等,该菌株对棉铃虫和豆青虫的致病率分别仅为约52%和8%。另一方面,由于该菌株在棉铃虫和豆青虫之间的致死率也差异显著,对其进一步的分析表明:其基因表达的特征主要是与表皮降解和菌株的定殖有关,对棉铃虫的响应中有许多相关表皮降解和定殖的基因均为显著上调表达,而两者间相关涉及黏附基因（Mad1）、过敏原蛋白（Bb-f2）和III型几丁质酶（Bbchi-17）的表达则无显著差异。除Bb-f2在球孢白僵菌响应非原始宿主时显著上调表达外,转录组数据的分析还显示菌株的一些其它过敏原相关基因rasp f 7 allergen和allergen V5/Tpx-1family protein也是上调表达的（不在Top 20）,推测Bb-f2等过敏原基因在菌株中的高量表达可能是不同宿主环境胁迫的结果。本研究差异表达基因的组学分析揭示了球孢白僵菌感染不同宿主致病性差异的分子机理,同时表明:球孢白僵菌某一特定菌株侵染不同昆虫宿主时,其代谢响应和感染策略会发生相应的变化以适应新的宿主,感染适应性的关键因素之一是需要去突破不同昆虫表皮几丁质组分的屏障及抵抗来自不同宿主的免疫压力。3.球孢白僵菌过敏原蛋白Bb-f2基因的荧光定量表达检测Bb-f2基因在球孢白僵菌响应敏感宿主家蚕时低表达,响应非原始分离宿主棉铃虫和豆青虫表皮时高表达,为了验证Bb-f2基因在其它非原始分离宿主中的表达模式,另增加检测了4种昆虫（2种鳞翅目昆虫大蜡螟和斜纹夜蛾,2种鞘翅目昆虫黄粉虫和大麦虫）被选择用于测试Bb-f2基因的表达。菌株GXsk1011对大蜡螟、斜纹夜蛾、黄粉虫和大麦虫的致病性不同,死亡率分别为46.67%、50%、26.67%,不感染大麦虫。q RT-PCR的结果显示Bb-f2基因在菌株响应斜纹夜蛾、棉铃虫、大蜡螟、黄粉虫、豆青虫和大麦虫表皮时相比于家蚕表皮均为上调表达,Bb-f2在球孢白僵菌应对非原始宿主时均具有高表达的倾向,推测Bb-f2的高表达是菌株的一种免疫应变反应以更好地适应新的宿主环境。同一株球孢白僵菌侵染敏感和非原始分离宿主时,Bb-f2呈现出表达量的差异,那么不同的球孢白僵菌分离株侵染同一宿主家蚕时,Bb-f2是怎样的表达模式呢,本研究通过q RT-PCR进行了探索,5株对家蚕毒力不同的球孢白僵菌菌株(2株生防菌株,3株来自家蚕的不同区域的分离菌株,LC₅₀由小到大依次为家蚕分离菌株GXsk1011 4.2×10⁵、YNsk1106 4.8×10⁵、SCsk1006 5.4×10⁵及生防菌株GXtr1010 8.9×10⁷、GXtr1009 1.0×10⁸conidia/m L)的荧光定量结果显示Bb-f2基因在对家蚕毒力最高的球孢白僵菌GXsk1011中表达量最低,在对家蚕毒力最低的球孢白僵菌GXtr1009中表达量最高,这一结果也表明Bb-f2基因的表达与非原始宿主环境的免疫压力有关,推测Bb-f2基因的高表达有利于菌株自身的保护和生存。4.球孢白僵菌过敏原蛋白基因Bb-f2的功能研究与分析克隆Bb-f2基因并利用在线网站和序列分析软件对Bb-f2进行生物信息学分析,Bb-f2基因编码260个氨基酸,是一个稳定蛋白,在N端含有22个氨基酸的信号肽,具有4个潜在的N-糖基化位点,保守结构域分析发现,Bb-f2与Asp f 2过敏原蛋白具有类似的保守结构域,属于类M35蛋白超家族,但含有HRxx H的保守基序,一般不具有蛋白酶活性。对Bb-f2的保守结构域分析表明存在假定锌离子结合位点,推测可能有锌离子结合功能。通过基因同源重组置换及插入强启动子的方法,成功获得了基因的敲除突变株（ΔBbf2）和超量表达突变株（OE-Bb-f2）。构建的突变株在1/4萨氏培养基上培养,观察并检测敲除株和超量株的生长,结果表明:Bb-f2的敲除和超量表达对球孢白僵菌的生长速度均无显著影响;Bb-f2基因的超量表达能显著增加球孢白僵菌的菌丝耐热性,热处理不同时间段后的菌落直径下降率显著低于野生型菌株,但Bb-f2基因的缺失也能增加菌丝耐热性,暗示Bb-f2基因缺失可能激发了其它基因增强耐热作用;突变株和野生株对高渗透剂（1M Na Cl）、细胞壁抑制剂（500μg/m L刚果红Congo red）以及金属离子(2 m M Zn²⁺)的敏感性试验结果表明:ΔBb-f2敲除株对Na Cl、Zn Cl₂、刚果红的胁迫表现出较高的敏感性,但OE-Bb-f2超量菌株相对于野生株,其菌落形态和直径大小均无显著差异,即表现为不敏感。致病性实验中,表皮接种侵染5龄家蚕和4龄棉铃虫检测突变株的毒力,以野生株作为对照,Bb-f2基因的超量表达增强了菌株对家蚕和棉铃虫的毒力,但Bb-f2基因的缺失也会显著的增强菌株对家蚕和棉铃虫的毒力。由于菌株对家蚕的致死率为100%,以测定LT₅₀来比较菌株间的毒力,OE-Bbf2超量株对家蚕的LT₅₀为4.0±0.02 d相比野生型表现为降低,但ΔBb-f2菌株对家蚕的LT₅₀为3.9±0.02 d比野生型的LT₅₀（4.5±0.03 d）也表现为明显缩短;野生株、敲除株和超量株在1×10⁸conidia/m L下对棉铃虫的校正死亡率分别为55.3%、67.68%和59.77%,敲除株和超量株的毒力明显高于野生株。5.球孢白僵菌Bb-f2过敏原蛋白作用机制的探索本研究通过异源真核细胞表达Bb-f2蛋白并将蛋白注射家蚕的方式,检测了对Toll通路上相关家蚕免疫反应的影响,分析和探索了Bb-f2蛋白在球孢白僵菌侵染昆虫过程中的作用机制。通过能谱分析仪测定家蚕、棉铃虫和豆青虫表皮均含有锌离子,电感耦合等离子体原子发射光谱法（ICP-AES）测定表皮中锌元素含量棉铃虫皮（44.47μg/g）>蚕皮（30.09μg/g）>豆青虫皮（25.21μg/g）,表明Bb-f2的表达量高低与三种昆虫表皮锌元素含量不完全呈正相关关系,暗示可能还存在其它宿主的环境因子影响Bb-f2的表达。另一方面,在球孢白僵菌生长的蚕皮基本盐培养基中添加不同浓度的锌离子,造成锌胁迫环境,检测了5个与球孢白僵菌锌离子代谢相关的基因并随机筛选了其它4个基因的表达量的变化,在不同锌离子浓度条件下,上述基因的表达多数受到Zn²⁺的抑制,但在1μmol/L Zn²⁺低浓度条件下,Bb-f2基因和球孢白僵菌锌指蛋白（zinc finger protein,BBA＿05776）等则为上调表达,随着锌离子浓度的升高,呈现下调趋势,暗示低浓度锌胁迫时,Bb-f2和锌指蛋白的高表达可能是参与了锌离子的摄取捕获及锌离子相关代谢。通过真核毕赤酵母表达Bb-f2蛋白,体外锌离子结合实验证明Bb-f2具有锌离子结合活性,表明Bb-f2参与了真菌锌离子的相关代谢,锌元素能够造成相关基因表达量的差异但不是由锌元素含量的多少直接决定Bb-f2的表达量。q RT-PCR结果显示ΔBb-f2敲除株中锌指蛋白显著上调表达,超量表达株OE-Bbf2中锌指蛋白显著下调表达。锌指蛋白基因编码环形E3泛素转移酶,决定泛素化修饰中靶蛋白的特异性识别,可通过泛素化途径降解靶蛋白（如变性、过量表达及许多调控蛋白）。在Bb-f2基因敲除株中锌指蛋白表现为显著上调,推测此时可能转由锌指蛋白发挥其生物学功能以调控菌株表现出更强的毒力,Bb-f2的超量表达菌株则由Bb-f2自身承担主要角色,相比野生株也表现为对宿主的高致病性。分别注射Bb-f2蛋白于感染球孢白僵菌的家蚕组和球孢白僵菌灭活孢子免疫应激的家蚕组,以注射PBS和牛血清白蛋白为对照,试验结果显示:Bb-f2蛋白对家蚕感染球孢白僵菌组和灭活孢子免疫应激家蚕组的Toll信号通路具有相似的影响结果,Bb-f2蛋白抑制了家蚕模式识别蛋白β-葡聚糖识别蛋白Bmβgrp 2和Bmβgrp 4及Toll样受体Bmtoll 7的表达,导致了抗菌肽Bmlebocin 1和Bmgloverin 2显著下调表达,有利于菌株的生存和定殖。模拟超量表达即家蚕感染球孢白僵菌后注射Bb-f2蛋白的毒力试验结果,缩短了野生菌株对家蚕的半致死时间,表明Bb-f2基因通过高量表达去适应不同的宿主环境而表现为菌株毒力的增强。综上结果,从家蚕分离的高毒力球孢白僵菌菌株GXsk1011对棉铃虫和豆青虫表现为低致病性,其差异表达基因的组学分析表明,典型的毒力基因如黏附蛋白Mad1,蛋白酶Pr2等为显著性下调表达,但过敏原蛋白Bb-f2（BBA＿05395）和III型几丁质酶（BBA＿05353）等为显著上调表达,其中对Bb-f2进一步检测了在另外4种昆虫表皮响应的表达特征,Bb-f2均为上调表达;对Bb-f2基因功能的研究表明,该基因涉及菌株的耐热性、化学因子的敏感性及毒力的增强;Bb-f2蛋白能够影响家蚕的Toll信号通路,抑制宿主的部分抗菌肽表达,以增强菌株感染不同宿主的适应性。本研究对揭示球孢白僵菌感染不同宿主的致病性差异及感染适应性的分子机理具有重要的意义。更多还原

【Abstract】 Beauveria bassiana is an important entomopathogenic fungus,which the pathogenic process to insects can be divided into three stages: epidermis penetration,colonization and growth in the blood of insects,the hyphae re-penetration of the host surface,and reproduction from in vivo to in vitro.So far,a large number of related genes have been identified in each infection stage of B.bassiana,and the main goal is to improve the virulence of fungi and biological control.In the process of infection,B.bassiana can infect the host,whose range is wide,through the insect epidermis.However,B.bassiana isolated from a specific host shows significant differences in pathogenicity when infecting different host insects,the molecular mechanism of the pathogenicity difference and how to adapt to the host environment of different insects for B.bassiana are still unclear.The main purpose of this study is to find the genes related to the parasitic adaptive function of B.bassiana in the process of infecting different hosts.We analyzed the gene expression characteristics of B.bassiana in response to different hosts by comparative transcriptomics method,uncovered the molecular mechanism of different pathogenicity of the specific B.bassiana strain to different insect hosts in this study,and found some special and differential expression genes of B.bassiana adapting to different hosts,such as the allergen gene（Bb-f2,BBA₀5395）,Class III chitinase Chi A2 gene（Bbchi-17,BBA 05353）and other genes,the expression of allergen protein Bb-f2 of highly virulent strain（GXsk1011）isolated from Bombyx mori,in particular,was significantly up-regulated in response to the epidermis of other hosts.The biological functions and effects of the Bb-f2 gene werestudied by gene knockout and overexpression methods.The expression characteristics of pattern recognition receptors PRRS,Toll-like receptors TLR,cytoplasmic signal transduction factor and antimicrobial peptide genes in the Toll pathway of silkworm were detected by heterologous eukaryotic cells expressing Bb-f2 protein and injecting the protein into silkworms,the mechanism of Bb-f2 protein in the process of B.bassiana infecting insects were analyzed and explored.The main results are as follows:1.Pathogenicity of B.bassiana GXsk1011 to Bombyx mori,Helicoverpa armigera and Clanis bilineataWhen B.bassiana GXsk1011 was used to infect three lepidopteran insects,fifth instar of B.mori,H.armigera and C.bilineata at a concentration of 1 × 108 conidia /ml,the mortality rates of B.bassiana to B.mori,H.armigera and C.bilineata were 100 %,52.50 % and 8.88 % respectively;the LC50 of B.mori,H.armigera and C.bilineata were successively 1.25 × 106,4.67 × 107 and 5.25 × 1010 conidia /ml.The results showed that the virulence of B.bassiana to the originally isolated（susceptible）host B.mori was significantly different from that of the two non-originally isolated hosts.2.Differentially expressed genes in response of B.bassiana to three lepidopteran insectsTo reveal the reasons for the differences in pathogenicity,the transcriptome data of B.bassiana responding to three insect epidermis were analyzed.Due to the low biomass of B.bassiana in the early infection stage at 24 h,and to avoid the digestion of RNase in living tissues and the influence of other microorganisms,in vitro simulation infection test and RNA-seq were used,and the transcriptome data were registered by the laboratory in the NCBI database（registration number: SRR3036777,SRR 3043073 and SRR 3043097）.The analysis of transcriptome data showed that,through pairwise comparison,there were about 600 to 900 genes of up-regulated expression and 400 to 800 genes of down-regulated expression.Through the annotation of differential genes,the differentially expressed genes mainly involving the biological processes and pathways of metabolism,catalytic activity,transporter activity and localization of strains,some key virulence genes closely related to pathogenicity differences and gene expression patterns of strains responding to different host epidermis were identified.The analysis of the top 20 differentially expressed genes with the highest multiple of difference（Top 20）showed that the key factors involved in spore wall adhesion,cuticleprotein degradation and some growth genes were significantly up-regulated expression in the infection process when B.bassiana responded to the sensitive host B.mori,such as adhesion protein（Mad1）,trypsin-like protease（Pr2）,O-methyltransferase,family 3（BBA₀9875）,cell surface protein（BBA₀9174）and so on,besides the Top 20 genes,endothiapepsin-like aspartate protease gene（Bbenp L-1）,which can degrade the epidermis,was also up-regulated expression,due to the high expression of these virulence genes,the strain has high infectivity（100% mortality）to B.mori;however,when B.bassiana responded to the non-original hosts of H.armigera and C.bilineata,the genes significantly up-regulated were allergen protein major allergen Asp f 2-like protein（Bb-f2,BBA₀5395）,Class III chitinase Chi A2（Bbchi-17,BBA₀5353）,nonribosomal peptide synthase（BBA₀8424）and so on,therefore,the pathogenicity to H.armigera and C.bilineata were only about 52 % and 8 % respectively.Additionally,due to the significant differences in mortality between H.armigera and C.bilineata,the further analysis showed that the gene expression characteristics of the strain were mainly related to epidermis degradation and fungal colonization,in response to H.armigera,the expressions of many epidermis degradations and colonization genes were significantly up-regulated,while the expressions of adhesion gene（Mad1）,allergen protein（Bb-f2）,Class III chitinase（Bbchi-17）showed no significant difference in terms of the two aspects.In addition to the significant up-regulation of Bb-f2 expression in B.bassiana responding to non-original hosts,transcriptome analysis also showed that some other allergens related genes rasp f7 allergen and allergen V5 / tpx-1 family protein were also up-regulated（not among the top 20）,it is speculated that the high expressions of Bb-f2 and other genes in the strain may be the result of different host environment stress.This study revealed the molecular mechanism of pathogenicity difference of B.bassiana infection in different hosts through the genomic analysis of differentially expressed genes,and also showed that when a specific strain of B.bassiana infects different insect hosts,its metabolic response and infection strategy will change accordingly to adapt to the new host,and one of the key factors to the infection adaptability is to break through the chitin barrier of different insect epidermis and resist the immune pressure from different hosts.3.Quantitative Real-time PCR detection of Bb-f2 allergen gene in B.bassianaThe Bb-f2 gene was lowly expressed when B.bassiana responded to sensitive hostB.mori and was highly expressed when responded to non-original isolated hosts H.armigera and C.bilineata epidermis,to verify the expression pattern of Bb-f2 gene in other non-original hosts,four additional insects（two Lepidoptera insects,Galleria mellonella and Spodoptera litura,two Coleoptera insects,Tenebrio molitor and Zophobas atratus）were selected to detect the expression of Bb-f2 gene.The pathogenicity of strain GXsk1011 to Galleria mellonella,Spodoptera litura and Tenebrio molitor was different,and the mortality rates were 46.67%,50% and 26.67%,respectively,non-infected with Zophobas atratus.The q RT-PCR results showed that the expression of the Bb-f2 gene,compared with responding to B.mori epidermis,was upregulated when the strains responded to the epidermis of Spodoptera litura,H.armigera,Galleria mellonella,Tenebrio molitor,C.bilineata and Zophobas atratus,it was speculated that Bb-f2 may have a tendency of high expression when B.bassiana respond to non-original host,and high expression of Bb-f2 is a kind of immune response for the strain to better adapt to a new host environment.When the same strain of B.bassiana infects sensitive and non-original hosts,the expression level of Bb-f2 is different,and what is the expression pattern of Bb-f2 when different strains of B.bassiana infect the same host B.mori? This study discussed this situation through q RT-PCR,the Quantitative Real-time results of 5 B.bassiana strains（2 biocontrol strains,3 separated strains from different areas of B.mori,the order of LC50 from small to large was B.mori-separated strains GXsk1011 4.2×105,YNsk1106 4.8×105,SCsk1006 5.4×105 and biocontrol strains GXtr1010 8.9×107,GXtr1009 1.0×108 conidia /m L）showed that the expression of Bb-f2 gene was the lowest in B.bassiana GXsk1011 with the highest virulence to B.mori,and the highest in B.bassiana GXtr1009 with the lowest virulence to B.mori,the results also suggested that the expression of Bb-f2 gene was related to the immune pressure of the non-original hosts,which can be speculated that the high expression of Bb-f2 gene was beneficial to the protection and survival of the strains themselves.4.Functional research and analysis of Bb-f2 allergen gene of B.bassianaCloning of Bb-f2 gene and Bioinformatics analysis of the Bb-f2 gene by using online website and sequence analysis software,Bb-f2 gene encodes has 260 amino acids,which is a stable protein and contains 22 amino acids in the N-terminal signal peptide and has four N-glycosylation sites,and conserved domain analysis showed that Bb-f2 and Asp f2-like allergen protein have similar conserved domains,belonging toM35-like protein superfamily,however,which contains the conserved motif of HRxx H and generally has no protease activity.The analysis of the conserved domain of Bb-f2 showed that it has a putative zinc ion binding site,which may be speculated to have a zinc ion binding function.Through homologous recombination replacement and strong promoter insertion,the gene knockout（ΔBb-f2）and overexpression mutant（OE-Bb-f2）were successfully obtained.The constructed mutant was cultured on 1/4 Sabouraud’s medium,and the growth of the knockout and overexpression strains were observed and detected,the results showed that the knockout and overexpression of Bb-f2 had no significant effect on the colony growth rate of B.bassiana;overexpression of Bb-f2 gene could significantly increase the mycelia heat tolerance of B.bassiana,and the decrease rate of colony diameter was significantly lower than that of wild-type strain after heat stress for different periods,however,deletion of Bb-f2 gene could also increase the heat tolerance of mycelia,suggesting that deletion of Bb-f2 gene might stimulate other genes to enhance heat tolerance;the results of the sensitivity test for the mutant and wild strain to high permeability（1m Na Cl）,cell wall inhibitor（500 μg / ml Congo red）and metal ions（2 m M Zn2+）showed that: knockout strain Δ Bb-f2 was highly sensitive to Na Cl,Zn Cl2 and Congo red stress,but overexpression strain OE-Bb-f2 was not significantly different from wild strain in colony morphology and diameter,meaning,it was not sensitive.In the pathogenicity test,the pathogenicity of the mutant was tested by cuticle inoculation with B.mori（5th instar）and H.armigera（4th instar）,and the wild strain was used as a control,the overexpression of the Bb-f2 gene enhanced the virulence of the strain to B.mori and H.armigera,however,the knockout of the Bb-f2 gene also significantly enhanced the virulence of the strain to B.mori and H.armigera.Because the lethal rate of the strain to silkworm was 100 %,the virulence of the strain was compared by measuring LT50,the LT50 of overexpression strain OE-Bb-f2 was 4.0 ± 0.02 days,which was lower than the that of the wild type,but the LT50 of ΔBb-f2 strain to B.mori was 3.9 ± 0.02 days,which was also significantly shortened than the LT50 of wild type（4.5 ± 0.03 d）;when the concentration of wild strain,knockout strain and overexpression strain was 1 × 108 conidia /m L,the corrected mortality of H.armigera was 55.3%,67.68 % and 59.77 % respectively,the virulence of knockout strain and overexpression strain was higher than that of wild strain.5.Study on the mechanism of action of Bb-f2 allergen protein of B.bassianaIn this study,expression of Bb-f2 protein in heterologous eukaryotic cell and injecting the protein into B.mori was used to detect the effect on the immune response of B.mori related to Toll pathway,the mechanism of Bb-f2 protein in the process of B.bassiana infecting insects were analyzed and explored.The contents of zinc in the epidermis of B.mori,H.armigera and C.bilineata were determined by the energy spectrum analyzer,the contents of zinc in the epidermis of H.armigera（44.47 μg / g）> B.mori（30.09 μg / g）> C.bilineata（25.21 μg / g）was determined by inductively coupled plasma atomic emission spectrometry（ICP-AES）,which demonstrated that high and low expression levels of Bb-f2 were not positively correlated with the cuticle zinc element contents in three insects,it suggested that there may be other host environmental factors affecting the expression of Bb-f2.On the other hand,different concentrations of zinc ions were added to the basic salt medium of B.mori epidermis grown by B.bassiana to create a zinc stress environment,the expression changes of five zinc-related genes and four randomly selected genes were detected,the gene expressions were generally inhibited by Zn2+,at 1 μmol /L Zn2+ concentration,the expression of Bb-f2 gene and zinc finger protein（BBA 05776）of B.bassiana were simultaneously up-regulated,which showed a downward trend with the increase of zinc ion concentration,it is suggested that when at a low concentration of zinc stress,the high expression of Bb-f2 and zinc finger protein might be involved in zinc uptake and zinc metabolism.Through the expression of Bb-f2 protein in eukaryotic Pichia pastoris,together with in vitro zinc ion binding experiment,it was indicated that Bb-f2 has zinc-binding activity and was indicated that Bb-f2 involved in the fungal zinc ions metabolism,it is suggested that zinc can cause the differences of expression,but the differences are not directly determined by the zinc content.q RT-PCR results showed that the expression of zinc finger protein was significantly up-regulated in ΔBb-f2 knockout strain and down-regulated in OE-Bb-f2 overexpression strain.Zinc finger protein gene encodes circular E3 ubiquitin transferase,which determines the specific recognition of target proteins in ubiquitination modification,can degrade target proteins（such as denaturation,overexpression and a great many regulatory proteins）through the ubiquitination pathway.In the Bb-f2 gene knockout strains,the expression of zinc finger protein was significantly up-regulated,which can be speculated that zinc finger protein may play its biological function toregulate the strain to show stronger virulence,and the overexpression strain of Bb-f2 was mainly caused by Bb-f2 itself,besides,compared with the wild type strain,it also showed high pathogenicity to the hosts.By injecting Bb-f2 protein into the B.mori infected with B.bassiana and the B.mori immunized activation with inactivated spores of B.bassiana,compared with PBS injection and BSA injection,the results showed that Bb-f2 protein had a similar effect on the Toll signaling pathway of B.mori infected with B.bassiana and the B.mori immunized activation with inactivated spores of B.bassiana,Bb-f2 protein inhibited the expression of pattern recognition proteins Bmβgrp 2,Bmβgrp 4 and Toll-like receptor Bmtoll 7 in the B.mori,lead to the antimicrobial peptides Bmlebocin 1 and Bmlebocin 2 in the B.mori were significantly down-regulated,which beneficial to the survival and colonization of the strain.After the B.mori was infected with B.bassiana,the injection of Bb-f2 protein through simulation overexpression shortened the half lethal time of B.mori,which indicated that the virulence of strain was enhanced by high-level expression of Bb-f2 gene to adapt to the different host environment.In conclusion,the highly virulent B.bassiana strain GXsk1011 separated from B.mori showed low pathogenicity to H.armigera and C.bilineata,and the analysis of the differentially expressed genes showed that the expressions of typical virulence genes such as adhesion protein Mad1 and protease Pr2 were significantly down-regulated,but the expressions of allergen protein Bb-f2（BBA₀5395）and type III chitinase（BBA₀5353）were significantly up-regulated,and the expression of Bb-f2 was upregulated after the expression characteristics of Bb-f2 in the epidermis response of the other four insects were further detected;The study for the function of Bb-f2 gene showed that the gene was involved in heat tolerance of strains,sensitivity of chemical factors and enhancement of virulence;In order to enhance the adaptability of the strain to different hosts,the Bb-f2 protein could affect the Toll signaling pathway of B.mori and inhibit the expression of some antimicrobial peptides in the hosts.This study is of great significance to reveal the pathogenicity difference of B.bassiana infection to different hosts and the molecular mechanism of infection adaptability.更多还原