球孢白僵菌两种甘露醇脱氢酶及五种过氧化氢酶的功能分析与对鳞翅目幼虫高口服毒力的工程菌株构建
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摘要
球孢白僵菌已被开发成多种制剂应用于农林害虫的防治,但其田间防治效果受环境因子的制约,这是制约真菌杀虫剂大规模应用推广的技术瓶颈和亟待解决的国际难题。开展白僵菌抗逆生物学研究,揭示抗逆性状的分子基础,定向遗传改良生产菌株的抗逆力和毒力,是提升生防真菌制剂质量和田间应用稳定性的核心技术途径。
甘露醇是生物体内广泛存在的小分子多元醇,作为碳源储备物,也参与真菌的抗逆境胁迫生理反应。过氧化氢酶是细胞抗氧化酶系的重要成员,在细胞抵抗活性氧自由基(Reactive oxygen species, ROS)胁迫过程中发挥着重要的作用。目前在生防真菌中有关甘露醇代谢相关酶系的生物学功能知之有限,对过氧化氢酶在抗逆反应中的作用也鲜有研究报道。另外,将外源苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)营养期杀虫蛋白Vip3Aa1基因置于构巢曲霉组成型通用启动子PgpdA下导入球孢白僵菌中能有效提高工程菌株对斜纹夜蛾低龄幼虫的毒力,但受启动子驱动力的影响,Vip3Aal毒蛋白的表达量不足以致死高龄的叶面暴食性害虫。为此,本论文从揭示球孢白僵菌抗逆生物学机理及提高菌株口服毒力的角度出发,运用农杆菌介导的同源重组以及芽生孢子化学转化法,系统研究了球孢白僵菌中甘露醇代谢相关酶和过氧化氢酶的基因家族的时空表达情况以及各基因缺失对菌株抗逆力和毒力的影响；以球孢白僵菌自身疏水蛋白基因启动子的克隆及优化为切入点,构建了高表达Vip3Aa1蛋白的球孢白僵菌工程菌株,并评价了其对斜纹夜蛾各龄幼虫双途径感染的杀虫活性。主要研究内容及结果如下：
甘露醇1-磷酸脱氢酶(mannitol1-phosphate dehydrogenase, MPD)的基因克隆、表达纯化及酶学特征分析通过序列比对及简并引物扩增,从球孢白僵菌Bb2860基因组中克隆到甘露醇1-磷酸脱氢酶基因BbMPD,序列全长1334bp,其开放阅读框(open reading frame, ORF)长1176bp,含一个158bp的内含子,编码一条由391个氨基酸组成的典型的真菌甘露醇1-磷酸脱氢酶,预测分子量为42.8kDa,与已知的其它17种真菌MPD蛋白的序列同源性为56～74%,且在N端含有一个NADH辅因子结合域(GAGRIG)。在大肠杆菌中成功表达了BbMPD的重组蛋白,其纯化产物的最适反应温度为30℃,最适反应pH7.0,对果糖6-磷酸具有底物专一性,米氏常数Km和Vmax积分别为0.89±0.05mM和913.5±14.9U/mg蛋白,还原D-果糖6-磷酸的催化效率(kcat/Km)为1.31×104mM-1s-1。
甘露醇脱氢酶(mannitol dehydrogenase, MTD)的基因克隆及两个脱氢酶基因的表达调控首次从Bb2860基因组中克隆到甘露醇脱氢酶基因BbMTD,其上游序列有3个胁迫响应元件(AGGGG)和5个碳利用阻遏元件CREA (Carbon catabolite repression element, SYGGRG)附着位点的保守序列；ORF全长801bp,编码266个氨基酸,蛋白分子量和等电点分别为28.2kDa和6.6,与其它真菌MTD的同源性较高,N端第28个氨基酸处存在一个NADPH辅因子结合域(GPKGIG)。
BbMTD在菌丝生长的前五天转录表达水平逐步递增,第五天的表达量约为第二天的20倍,但在此后的两天里又有所下降。BbMPD的转录水平在连续七天培养过程中保持相对恒定,第4天表达水平最高。在1M NaCl的渗透压胁迫和50mM H202的氧化胁迫下,基因表达均呈现随胁迫时间延长而显著上调的趋势,渗透压胁迫3h后的BbMTD和BbMPD基因表达水平分别较空白对照提高了5.0和10.9倍；相应基因的转录水平在氧化胁迫3h后分别上调了11.4和74.9倍。两基因在热胁迫条件下的表达水平均受抑制,热激3h后,BbMPD的表达水平为正常条件下的63%,而BbMTD只有一半左右。结果表明,两基因均参与球孢白僵菌抵抗逆境胁迫的防御反应。
BbMPD和BbMTD基因的敲除及突变株表型分析应用根癌农杆菌介导的遗传转化方法成功构建BbMPD和BbMTD基因的缺失突变株ΔBbMPD和ABbMTD。酶活分析显示,BbMPD和BbMTD均是球孢白僵菌甘露醇代谢的关键催化酶,ΔBbMPD菌株粗酶液无法还原果糖6-磷酸,而ABbMTD有低水平的酶活残留。无论是在固体SDAY平板正常条件下生长,还是在液体SDB胁迫条件下培养,单基因敲除株胞内甘露醇含量均显著低于野生株。在正常生长条件下,ΔBbMPD和ΔBbMTD菌丝(或分生孢子)中甘露醇的含量分别减少了68%(83%)和16%(38%)。渗透压能诱导菌株胞内甘露醇的合成,1M NaCl刺激24h后,ΔBbMPD和ΔBbMTD菌丝甘露醇含量分别提高3.0和1.7倍。而热激培养则抑制其合成,35℃下培养一天,相应的甘露醇合成水平只有正常培养条件下的40.7%和62.3%。有趣的是,各菌株胞内甘露醇合成的减少伴随着海藻糖含量的上升。例如,正常平板培养条件下,ΔBbMPD菌丝和分生孢子中海藻糖的含量较野生株分别提高1.7倍和1.5倍。
除胞内可溶性甘露醇和海藻糖含量变化外,BbMPD基因的敲除显著影响菌株的产孢能力(下降27%),而ΔBbMTD菌株的产孢则不受影响。两敲除株菌丝和孢子在不同的单一碳源培养基上培养时表型差异明显。ΔBbMPD菌丝生长速度不受影响,但分生孢子的萌发速率较野生株显著降低,在以果糖,葡萄糖和甘露醇为单一碳源平板上孢子萌发50%所需的时间(GT50)分别延迟3.0、3.1和6.6h；ΔBbMTD菌丝生长则受到显著抑制,但其分生孢子除在甘露醇平板上萌发延迟之外(GTso后延7.1h),萌发速率在其他碳源平板上差异不显著。抗逆力测定显示,甘露醇合成受阻能致球孢白僵菌的分生孢子对活性氧、高温、紫外等环境胁迫的适应能力显著下降。ΔBbMPD和ΔBbMTD分生孢子对H202的耐受力分别较野生株下降38%和18%,对UV-B辐射的耐受力(LDso：0.26和0.36J/cm2)比野生株(LD50：0.42J/cm2)分别下降39%和16%,对45℃热胁迫的耐受力(LT50：39.0min和46.1min)比野生株(52.0min)分别下降22%和11%。然而,上述基因的缺失不显著影响菌株对桃蚜的毒力以及对高渗透压胁迫的耐受力。
球孢白僵菌过氧化氢酶(Catalase,CAT)基因家族的克隆、序列分析及表达调控通过保守序列比对从球孢白僵菌2860基因组中克隆到五个CAT基因,分别编码三种单功能CAT (BbcatA、 BbcatB和BbcatC)和两种双功能CAT (BbcatP和BbcatD)。其中,BbcatC为小亚基酶,蛋白序列中含有NADPH绑定域,其余四种均为大亚基酶。通过进化树构建分析表明,五种CAT分别属于不同的蛋白家族。其中,BbcatA属于cladeA(孢子特异性CAT),主要参与分生孢子的形成及发育过程,其基因转录表达水平在产孢过程中逐渐累积,而在孢子萌发和菌丝开始生长过程中大幅降低；氧化胁迫能提高该基因转录水平,50mM H202胁迫3h的表达水平为空白对照的4.5倍。BbcatB和BbcatD属于clade B(分泌性CAT),在蛋白N端均含有一段19个氨基酸的分泌信号肽。BbcatB基因的表达水平随菌落生长时间延长而逐渐上调,第7天的转录水平达到第二天的30.2倍。BbcatD基因的表达在生长的前5天内保持较低的水平,而在第6天和第7天显著增强,分别达到第二天的53.1和158.3倍。氧化胁迫均能诱导BbcatB和BbcatD基因的表达,分别提高8.6和6.9倍。BbcatC和BbcatP均属于clade P(过氧化物酶体CAT),它们分别在蛋白中间段和C端含有PTS信号序列。BbcatC基因在菌丝生长过程中的转录表达量逐渐上升,而BbcatP基因在7天的菌落生长过程中其表达水平相对平稳。与其他四种CAT基因的表达水平相比,BbcatP基因受H202胁迫的影响最为显著,3h胁迫处理后,其转录水平较正常条件提高13.5倍。结果表明,各CAT在球孢白僵菌生长发育过程中扮演不同的角色,且不同程度地参与氧化胁迫的防御反应。
球孢白僵菌五种过氧化氢酶的生物学功能分析应用根癌农杆菌介导的遗传转化方法成功构建球孢白僵菌各CAT基因的单敲除株。酶活测定显示,正常生长条件下,菌丝CAT活力主要取决于BbcatB、 BbcatP和BbcatD。活性染色PAGE凝胶上仅呈现BbcatB(高)和BbcatP(低)两条酶带,氧化胁迫及热激等胁迫条件并不能改变相应的酶谱类型。抗逆力测定表明,ΔBbcatC和ΔBbcatP菌丝对氧化胁迫十分敏感,50mM H202几乎完全抑制.ΔBbcatP菌落的生长,而其它三种CAT的缺失突变株的菌落生长没有受到明显抑制。ABbcatB菌落生长在含4mM甲萘醌的培养基上明显受抑制,其他CAT基因缺失株生长则差异不显著。在渗透压(NaCl)、杀菌剂(多菌灵)以及热胁迫(33℃)条件下,各敲除株的菌落生长表型均未受到显著影响。另一方面,CAT基因敲除后可显著影响分生孢子对H202、UV-B和高温的耐受力,但影响程度不同。ΔBbcatA、 ΔBbcatP和ΔBbcatC分生孢子对H202的耐受力分别较野生株下降42.6%、57.0%和34.5%,而ΔBbcatB和ΔBbcatD的分生孢子对此氧化胁迫的耐受力未受显著影响。各敲除株的孢子对UV-B辐射的耐受力也表现不同程度的下降,以ΔBbcatB和ΔBbcatD最为显著,较野生株分别下降52.4%和47.6%。耐热力测定显示,ΔBbcatA分生孢子在45℃热胁迫下的半致死时间LT50较野生株下降40%,但其余敲除株都与野生株的LT5o均为52.0min。
毒力测定显示,敲除株ΔBbcatA、 ABbcatP知ΔBbcatD对斜纹夜蛾二龄幼虫的毒力显著低于野生株,其LT50分别较野生株下降1.4天、1.8天和0.9天,而另两个敲除株与野生株无异,其LT5o为5.0～5.1天之间。由此可见,CAT是球孢白僵菌克服昆虫寄主ROS相关的抗氧化酶系,因而是影响菌株重要抗逆性状和毒力的重要因子。
球孢白僵菌内源强启动子Phydl的克隆与优化为了寻找一种可在球孢白僵菌中高表达外源有益基因的内源强启动子,从Bb2860基因组中克隆到I型疏水蛋白基因(hydl)的上游序列,并分别截成-1798(全长、-1290、-1179、-991和-791bp)不同长度的片段或对-1290片段特定转录因子(StuA、 NIT2和Mat-Mc)结合位点施以定点突变后,与绿色荧光蛋白报告基因eGFP融合起来,在球孢白僵菌Bb2860中转化表达,考察各转化子中eGFP蛋白相对于外源通用启动子PgpdA调控的表达水平。结果显示,携带3个必要转录因子的-1290bp为最优的启动子片段(Phydl-t1),由其驱动eGFP表达的转化子菌落的相对荧光强度(RFI)是PgpdA驱动的15.6倍,且eGFP蛋白主要积累于分生孢子形成阶段,尤以成熟的分生孢子中表达量最高。其他截头片段或定点突变的启动子片段启动基因表达的效率均不如Phydl-t1,-791bp片段几乎完全失去启动外源基因表达的能力。在定点突变的片段中,以StuA结合位点的突变影响eGFP基因表达最甚。
高表达中肠特异性杀虫蛋白Vip3Aa1的球孢白僵菌工程株的构建及毒力分析将外源Bt营养期杀虫蛋白基因Vip3Aa1置于优化的内源启动子Phydl-t1下导入野生株Bb2860中,筛选鉴定出一株生长和产孢性状良好且遗传稳定的工程株BbHV8。实时定量PCR和ELISA分析显示,与BbV28菌株(本实验室先前构建由PgpdA启动Vip3Aa1表达的工程菌株)相比,BbHV8培养4天的菌落(菌丝)中Vip3Aa1基因的转录水平上升112倍,在菌丝和培养7天形成的分生孢子中Vip3Aa1蛋白的表达量分别提高7.8和9.8倍。Western杂交和免疫胶体金定位分析表明,Vip3Aa1蛋白在BbHV8分生孢子中高表达且均与分布于细胞质中。孢子被四龄斜纹夜蛾幼虫摄食24h后,能在中肠液中通过Western杂交检测到一条62kDa的活性条带。生测实验表明,BbHV8菌株对斜纹夜蛾二至五龄幼虫均表现出明显的Vip3Aal特有的口服感染毒力,致死虫尸严重萎缩,保湿培养后长出的菌丝稀薄甚至不长。时间-剂量-死亡率(TCM)观察显示,BbHV8的杀虫速度明显快于BbV28和Bb2860,高浓度处理下,BbHV8在处理后第三天能分别杀死100%和53%的二、三龄幼虫,而BbV28和Bb2860在第8天才杀三龄幼虫62%和45%。
TCM模拟分析显示,Bb2860、BbV28和BbHV8对斜纹夜蛾二龄幼虫的LC5o随接种天数差异显著。BbHV8对斜纹夜蛾二龄幼虫的LC5o及其95%置信限由第2天的1293(889～1880)孢子/mm2下将至第5天的26(16～44)孢子/mm2。Bb2860和BbV28对三龄幼虫LC5o的差异不显著,但均显著高于BbHV8的LC5o。基于各菌株的LC5o值,BbHV8处理后3-8天期间对二、三龄幼虫的毒力分别为BbV28的8-80倍和Bb2860的18～931倍,毒力差异倍数随处理后时间延长而缩小。在可比较的处理剂量下,BbHV8对二、三龄幼虫的LTso比BbV28和Bb2860分别缩短1.5～3.8天和2.9～4.6天,即在给定剂量下,BbHV8的杀虫速度比BbV28快63～100%,比Bb2860怏1.1～1.6倍。此外,BbHV8在处理后3-8天杀死90%二至五龄幼虫的孢子剂量为678～1389个孢子/mm2,BbV28和Bb2860对三龄幼虫无可计算的LC5o。以上结果表明,Phydl-t1能强启动外源毒素基因的表达并大量积累毒蛋白于球孢白僵菌分生孢子中,从而大幅提高其对高龄暴食性害虫的毒力。
综上所述,本研究主要成果和创新点在于,一是成功克隆了球孢白僵菌中与甘露醇代谢相关的BbMPD和BbMTD基因,通过基因敲除和表型分析首次揭示了它们在调控该菌生长发育、抗逆及毒力性状中的重要作用。二是首次系统研究了该菌CAT基因种类、结构和功能,阐明了各CAT影响该菌抗逆性状和毒力的程度。三是发现了能驱动外源基因在球孢白僵菌分生孢形成阶段特异性超表达的疏水蛋白启动子Phyd1-t1,并利用其构建了超高表达Vip3Aa1肠毒蛋白的工程菌株,大幅提高了对斜纹夜蛾各龄幼虫的口服毒力,因而是对付暴食性叶面害虫的新武器。这些结果深化了对丝孢类生防真菌抗逆分子机理的认识,形成了真菌杀虫剂遗传改良的关键技术和成果。
As a classic fungal entomopathogen, Beauveria bassiana has been developed into dozens of mycoinsecticides and widely applied in insect pest control. The fungal application is often restricted due to the defect of slow action and the sensitivity to the stresses of high temperature and solar UV irradiation often encountered in summer. Thus, there is an urgent need for exploring possible mechanisms involved in the defence of the fungal pathogen against multiple stresses and improving genetically fungal virulence to target pests.
Mannitol, a small sugar polyol, is widely distributed in fungi and can be served as carbohydrate resources and involved in crucial defense mechanisms that protect cells from damages by environmental stresses. Catalases (CAT) are important antioxidant enzymes in eukaryotic cells and play important roles in protecting cells from the damage of intracellular reactive oxygen species (ROS). However, the regulation of mannitol metabolism and the function of catalases in entomopathogenic fungi are not well understood at present. On the other hand, integrating Bacillus thuringiensis (Bt) vegetative insecticidal protein Vip3Aal into B. bassiana under a constitutive promoter PgpdA from Aspergillus nidulans could increase fungal per os virulence to the neonates of Spodoptera litura but the amount of the target toxin expressed in fungal conidia was too small to kill older larvae. Thus, the present study sought to elucidate a possible mechanism involved in mannitol metabolism and explore the functions of CAT members in B. bassiana in response to multiple stresses by means of gene knockouts and phenotypic analyses. Moreover, a fragment found from the upstream regions of B. bassiana Class I hydrophobin gene (hydl) was used a novel, strong promoter to drive high yield expression of the midgut toxin Vip3Aal in transgenic B. bassiana, yielding an engineered strain highly insecticidal to all instars of S. litura larvae. The results are summarized below.
Characterization of B. bassiana mannitol1-phosphate dehydrogense (BbMPD). A full-length1334-bp fragment of mannitol1-phosphate dehydrogenase gene (BbMPD) was cloned from B. bassiana2860(Bb2860herein), including a putative1176-bp open reading frame (ORF) with a158-bp intron. The gene was found encoding a391amino acid protein and showing56-74%sequence identity to MPDs known from other17fungi. The deduced protein was featured with a putative NADH cofactor binding site (GAGRIG) at the N-terminal. The gene was successfully expressed in E. coli BL21and the recombinant protein (84.4kDa) was well in agreement with the predicted molecular weight. The purified BbMPD showed high substrate specificity to fructose6-phosphote (Fru6P). Its maximal activity in Fru6P reduction was achieved at30℃and pH7.0. In the reaction at optimal 30℃and pH7.0, the BbMPD activities (y) at0.05-5mM Fru6P (x) were well fitted to non-linear Michaelis-Menten equation (r2=0.99), generating the parameters Km and Vmax (±SE) of0.89±0.05mM and913.5114.9U/mg proteins. The catalytic efficiency (kcat/Km) for the reduction of Fru6P by BbMPD was estimated as1.31×104mM-1s-1.
Gene cloning of B. bassiana mannitol dehydrogense (BbMTD). A full-length864-bp fragment of mannitol dehydrogenase gene(BbMTD) was cloned from Bb2860. Three potential stress-responsive elements (AGGGG) and five elements meeting the consensus sequence (SYGGRG) for binding CREA, a broad domain regulator for carbon catabolism repression in Aspergillus, were lcoated in its5'flanking regions. The864-bp ORF with a63-bp intron encoded for a266amino acid protein with the predicted molecular weight of28.2kDa and the isoelectric point of6.6. The deduced protein, featured with a putative NADPH cofactor binding site (GPKGIG) at N-terminal, was found sharing high sequence identity to MTDs known from other fungi.
Expression patterns of BbMPD and BbMTD. The wild-type strain Bb2860was grown on the plates of Saubouraud dextrose agar plus1%yeast extract (SDAY) for7days at25℃to monitor the transcription levels of the two genes in fungal colonies. As a result, the mRNA level of BbMTD increased steadily during the first four-day growth, followed by a20-fold increase on day5relative to day2. Unlike BbMTD, the transcription level of BbMPD fluctuated in a narrow range during the7-day growth and peaked on day4. Under the osmotic stress of1M NaCl) or the oxidative stress of50mM H2O2, the expression levels of both genes increased with stress time. Three-hour stress resulted in an increase of5(NaCl) or11.4(H2O2) fold in BbMTD expression and of10.9(NaCl) and74.9(H2O2) fold in BbMPD expression, respectively. In contrast, both BbMPD and BbMTD expression levels were suppressed by50and63%, respectively, after3-h heat stress at35℃. The results suggest that both genes take important parts in B. bassiana response to multiple stresses.
Functional analyses of BbMPD and BbMTD. Two gene disruption mutants, ABbMPD and ABbMTD, were constructed via Agrobacterium tumefaciens-mediated transformation. Enzymatic activity assays and polyol content analysis showed that both BbMPD and BbMTD were crucial enzymes for mannitol metabolism. The BbMPD disruption resulted in almost all loss of MPD activity to reduce fructose-6-phosphate while the BbMTD knockout led to～80%loss of MTD activity compared with the wild-type strain. The two mutants and Bb2860grown on SDAY plates or incubated in Saubourau dextroe broth (SDB) differed significantly in intracellular mannitol content. The mannitol contents in mycelia and conidia decreased68and83%for△BbMPD, and16and38%for△BbMTD, respectively, compared with the Bb2860counterparts. The mannitol contents of△BbMPD and ΔBbMTD were enhanced respectively by3.0and1.7fold after the SDB-cultured mycelia were transferred to fresh SDB containing1M NaCl for24-h osmotic stress or by40.7and62.3%after exposure to35℃. Accompanied by the decreases of mannitol contents, trehalose contents increased in ΔBbMPD and ΔBbMTD cells under normal or stressed conditions.
The conidial yield of ΔBbMPD on SDAY was significantly reduced by25-27%compared with similar yields of Bb2860and ΔBbMTD. The two mutants showed some differences in colony growth and spore germination on minimal medium with different carbon sources. ΔBbMPD was similar to Bb2860in colony size irrespective of a carbon source but fructose, glucose and mannitol delayed50%spore germination (GT50) for3.0,3.1and6.6h, respectively. In contrast, ΔBbMTD grew much more slowly than Bb2860and its GT50was delayed for7.1h by mannitol but not affected by fructose and glucose. Moreover, ΔBbMPD and ΔBbMTD conidia were38and18%less tolerant to the oxidative stress of H2O2,39and16%less tolerant to the stress of UV-B irradiation, and22and11%less tolerant to the wet-heat stress of45℃, respectively. However, both gene knockout mutants were not affected in conidial tolerance to osmotic stress and and virulence to the apterous adults of green peach aphid Myzus persicae.
Gene cloning, sequence analysis and expression patterns of B. bassiana catalases. Five catalase (CAT) genes were cloned for the first time from B. bassina, including BbcatA, BbcatB, BbcatP, BbcatC and BbcatD. These genes were found encoding three monofunctional catalases (BbcatA, BbcatB, and BbcatC) and two biofunctional catalase-peroxidases (BbcatP and BbcatD). Of those, BbcatC was a small subunit catalase found in B. bassiana and other four were all large subunit catalases. Phylogenetic analysis indicated that five catalases were classified to different groups. BbcatA, a Clade A catalase, is mainly involved in spore-specific processes, such as germination. Its transcription level increased during conidiation but significantly decreased during spore germination and hyphal growth. In SDB cultures including50mM H2O2for3-h oxidative stress, the transcription level of BbcatA was enhanced by4.5fold. BbcatB and BbcatD were Clade B catalases due to the presence of a secretive signal peptide of19amino acids at the N-terminal. The BbcatB gene transcribed increasingly during colony growth, resulting in a peak of30.2fold increase on day7relative to day2. The transcript level of BbcatD was relatively low during the first5-day growth and drastically increased53.1and158.3fold on days6and7, respectively. BbcatC and BbcatP were Clade P members (peroxisomal catalases) due to their peroxisome-targeting signal (PTS). BbcatC transcribed increasingly during colony growth while BbcatP transcibed at a level much lower than other four genes during7-day colony growth. The transcriptional levels of both genes were induced by H2O2added to SDB cultures for3-h oxidative stress, which, for example, increased BbcatP transcription by13.5fold. Our study revealed that the B. bassiana catalases may take different parts in regulating fungal response to oxidative stress.
Functional analyses of five B. bassiana catalases. The disruption mutants of five B. bassiana CAT genes, i.e., ΔBbcatA, ΔBbcatB, ΔBbcatC, ABbcatD and AbbcatP, were constructed by means of A. tumefaciens-mediated transformation. Enzymatic activity assays indicated that BbcatB, BbcatP and BbcatD primarily contributed to the CAT activities of the fungal species under normal conditions. In the profiles of non-denaturing polyacrylamide gels stained with ferricyanide, however, only two CAT-active bands were found in the wild-type strain grown under normal conditions. The upper and lower bands corresponded to BbcatB and BbcatP because they were absent in the ΔBbcatB and ΔbbcatP profiles, respectively.
ΔBbcatC and ΔBbcatP were very sensitive to the oxidative stress of50mM H2O2with their colony growth being greatly suppressed (e.g., nearly100%for AbbcatP). However, other three mutants showed the same response to the oxidative stress as the wild-type strain. All mutant and wild-type strains displayed no significant difference in colony growth on SDAY plates under the stress of1M NaCl,1μg/ml carbendazim or4mM menadione or incubated at33℃. Exceptionally, the colony growth of ABbcatB was suppressed on menadione-inclusive plates. Moreover, conidial tolerance to H2O2was reduced by42.6,57.0and34.5%for ΔBbcatA, ΔBbcatC and AbbcatP, respectively. The conidia of ΔBbcatB and ABbcatD were52.4and47.6%less tolelrance to UV-B irradiation than the wild-type strain, respectively. Conidial tolerance to the heat stress of45℃was similar for all wild-type and mutant strains except AbbcatA, whose median lethal time (LT50) was reduced by40%.
ΔBbcatA, ΔBbcatP and ΔBbcatD displated significantly reduced virulence to the second-instar larvae of S. litura compared to the wild-type strain. Their LT50estimates were delayed for1.4,1.8and0.9days, respectively. The same estimates of ABbcatB, ABbcatC and the wild-type strain fell in the narrow range of5.0-5.1days. Thus, the three catalases are potential virulence factors in B. bassiana.
Great elevation of fungal per os virulence to caterpillars by high yield expression of a midgut-acting toxin in transgenic conidia under a novel promoter. Beuaveria bassiana infects insect pests normally through host cuticle but hardly causes per os infection. We obtained a great progress in high yield expression of Bacillus thuringensis Vip3Aal, a midgut-acting toxin, in transgenic conidia for enhanced fungal virulence to caterpillars through spore ingestion. The toxin production relied on a homologous promoter (Phydl-t1) found to drive the toxin-coding gene expression specifically during conidiation. The gene transcription level in the4-day colonies of all examined transformants was3.4-112fold higher than that in a transgenic strain (BbV28) under the control of PgpdA, a heterologous promoter widely used for gene expression in fungi. The best transformant BbHV8produced9.8-fold-higher Vip3Aal yield in ripe conidia than BbV28. The release of active Vip3Aal into larval midguts from BbHV8conidia ingested within24h was4.4-fold more than that from BbV28. In the bioassays of spore suspensions sprayed onto S. litura larvae (for normal infection) and lotus leaf discs (for feeding), the LC50S of BbHV8against instars II and III on days2-5and3-8were reduced by11-24and8-80fold (declining with time), respectively, compared with the BbV28estimates. BbHV8killed90%instars II-V larvae within3-8days under the economic sprays of678-1389conidia/mm2whereas BbV28and wild-type strain could not kill the older larvae effectively. Conclusively, the high toxin yield in conidia enhanced greatly the toxin release into midguts, the fungal per os virulence to caterpillars and the potential of BbHV8for pest control.
甘露醇是生物体内广泛存在的小分子多元醇,作为碳源储备物,也参与真菌的抗逆境胁迫生理反应。过氧化氢酶是细胞抗氧化酶系的重要成员,在细胞抵抗活性氧自由基(Reactive oxygen species, ROS)胁迫过程中发挥着重要的作用。目前在生防真菌中有关甘露醇代谢相关酶系的生物学功能知之有限,对过氧化氢酶在抗逆反应中的作用也鲜有研究报道。另外,将外源苏云金芽孢杆菌(Bacillus thuringiensis,简称Bt)营养期杀虫蛋白Vip3Aa1基因置于构巢曲霉组成型通用启动子PgpdA下导入球孢白僵菌中能有效提高工程菌株对斜纹夜蛾低龄幼虫的毒力,但受启动子驱动力的影响,Vip3Aal毒蛋白的表达量不足以致死高龄的叶面暴食性害虫。为此,本论文从揭示球孢白僵菌抗逆生物学机理及提高菌株口服毒力的角度出发,运用农杆菌介导的同源重组以及芽生孢子化学转化法,系统研究了球孢白僵菌中甘露醇代谢相关酶和过氧化氢酶的基因家族的时空表达情况以及各基因缺失对菌株抗逆力和毒力的影响；以球孢白僵菌自身疏水蛋白基因启动子的克隆及优化为切入点,构建了高表达Vip3Aa1蛋白的球孢白僵菌工程菌株,并评价了其对斜纹夜蛾各龄幼虫双途径感染的杀虫活性。主要研究内容及结果如下：
甘露醇1-磷酸脱氢酶(mannitol1-phosphate dehydrogenase, MPD)的基因克隆、表达纯化及酶学特征分析通过序列比对及简并引物扩增,从球孢白僵菌Bb2860基因组中克隆到甘露醇1-磷酸脱氢酶基因BbMPD,序列全长1334bp,其开放阅读框(open reading frame, ORF)长1176bp,含一个158bp的内含子,编码一条由391个氨基酸组成的典型的真菌甘露醇1-磷酸脱氢酶,预测分子量为42.8kDa,与已知的其它17种真菌MPD蛋白的序列同源性为56～74%,且在N端含有一个NADH辅因子结合域(GAGRIG)。在大肠杆菌中成功表达了BbMPD的重组蛋白,其纯化产物的最适反应温度为30℃,最适反应pH7.0,对果糖6-磷酸具有底物专一性,米氏常数Km和Vmax积分别为0.89±0.05mM和913.5±14.9U/mg蛋白,还原D-果糖6-磷酸的催化效率(kcat/Km)为1.31×104mM-1s-1。
甘露醇脱氢酶(mannitol dehydrogenase, MTD)的基因克隆及两个脱氢酶基因的表达调控首次从Bb2860基因组中克隆到甘露醇脱氢酶基因BbMTD,其上游序列有3个胁迫响应元件(AGGGG)和5个碳利用阻遏元件CREA (Carbon catabolite repression element, SYGGRG)附着位点的保守序列；ORF全长801bp,编码266个氨基酸,蛋白分子量和等电点分别为28.2kDa和6.6,与其它真菌MTD的同源性较高,N端第28个氨基酸处存在一个NADPH辅因子结合域(GPKGIG)。
BbMTD在菌丝生长的前五天转录表达水平逐步递增,第五天的表达量约为第二天的20倍,但在此后的两天里又有所下降。BbMPD的转录水平在连续七天培养过程中保持相对恒定,第4天表达水平最高。在1M NaCl的渗透压胁迫和50mM H202的氧化胁迫下,基因表达均呈现随胁迫时间延长而显著上调的趋势,渗透压胁迫3h后的BbMTD和BbMPD基因表达水平分别较空白对照提高了5.0和10.9倍；相应基因的转录水平在氧化胁迫3h后分别上调了11.4和74.9倍。两基因在热胁迫条件下的表达水平均受抑制,热激3h后,BbMPD的表达水平为正常条件下的63%,而BbMTD只有一半左右。结果表明,两基因均参与球孢白僵菌抵抗逆境胁迫的防御反应。
BbMPD和BbMTD基因的敲除及突变株表型分析应用根癌农杆菌介导的遗传转化方法成功构建BbMPD和BbMTD基因的缺失突变株ΔBbMPD和ABbMTD。酶活分析显示,BbMPD和BbMTD均是球孢白僵菌甘露醇代谢的关键催化酶,ΔBbMPD菌株粗酶液无法还原果糖6-磷酸,而ABbMTD有低水平的酶活残留。无论是在固体SDAY平板正常条件下生长,还是在液体SDB胁迫条件下培养,单基因敲除株胞内甘露醇含量均显著低于野生株。在正常生长条件下,ΔBbMPD和ΔBbMTD菌丝(或分生孢子)中甘露醇的含量分别减少了68%(83%)和16%(38%)。渗透压能诱导菌株胞内甘露醇的合成,1M NaCl刺激24h后,ΔBbMPD和ΔBbMTD菌丝甘露醇含量分别提高3.0和1.7倍。而热激培养则抑制其合成,35℃下培养一天,相应的甘露醇合成水平只有正常培养条件下的40.7%和62.3%。有趣的是,各菌株胞内甘露醇合成的减少伴随着海藻糖含量的上升。例如,正常平板培养条件下,ΔBbMPD菌丝和分生孢子中海藻糖的含量较野生株分别提高1.7倍和1.5倍。
除胞内可溶性甘露醇和海藻糖含量变化外,BbMPD基因的敲除显著影响菌株的产孢能力(下降27%),而ΔBbMTD菌株的产孢则不受影响。两敲除株菌丝和孢子在不同的单一碳源培养基上培养时表型差异明显。ΔBbMPD菌丝生长速度不受影响,但分生孢子的萌发速率较野生株显著降低,在以果糖,葡萄糖和甘露醇为单一碳源平板上孢子萌发50%所需的时间(GT50)分别延迟3.0、3.1和6.6h；ΔBbMTD菌丝生长则受到显著抑制,但其分生孢子除在甘露醇平板上萌发延迟之外(GTso后延7.1h),萌发速率在其他碳源平板上差异不显著。抗逆力测定显示,甘露醇合成受阻能致球孢白僵菌的分生孢子对活性氧、高温、紫外等环境胁迫的适应能力显著下降。ΔBbMPD和ΔBbMTD分生孢子对H202的耐受力分别较野生株下降38%和18%,对UV-B辐射的耐受力(LDso：0.26和0.36J/cm2)比野生株(LD50：0.42J/cm2)分别下降39%和16%,对45℃热胁迫的耐受力(LT50：39.0min和46.1min)比野生株(52.0min)分别下降22%和11%。然而,上述基因的缺失不显著影响菌株对桃蚜的毒力以及对高渗透压胁迫的耐受力。
球孢白僵菌过氧化氢酶(Catalase,CAT)基因家族的克隆、序列分析及表达调控通过保守序列比对从球孢白僵菌2860基因组中克隆到五个CAT基因,分别编码三种单功能CAT (BbcatA、 BbcatB和BbcatC)和两种双功能CAT (BbcatP和BbcatD)。其中,BbcatC为小亚基酶,蛋白序列中含有NADPH绑定域,其余四种均为大亚基酶。通过进化树构建分析表明,五种CAT分别属于不同的蛋白家族。其中,BbcatA属于cladeA(孢子特异性CAT),主要参与分生孢子的形成及发育过程,其基因转录表达水平在产孢过程中逐渐累积,而在孢子萌发和菌丝开始生长过程中大幅降低；氧化胁迫能提高该基因转录水平,50mM H202胁迫3h的表达水平为空白对照的4.5倍。BbcatB和BbcatD属于clade B(分泌性CAT),在蛋白N端均含有一段19个氨基酸的分泌信号肽。BbcatB基因的表达水平随菌落生长时间延长而逐渐上调,第7天的转录水平达到第二天的30.2倍。BbcatD基因的表达在生长的前5天内保持较低的水平,而在第6天和第7天显著增强,分别达到第二天的53.1和158.3倍。氧化胁迫均能诱导BbcatB和BbcatD基因的表达,分别提高8.6和6.9倍。BbcatC和BbcatP均属于clade P(过氧化物酶体CAT),它们分别在蛋白中间段和C端含有PTS信号序列。BbcatC基因在菌丝生长过程中的转录表达量逐渐上升,而BbcatP基因在7天的菌落生长过程中其表达水平相对平稳。与其他四种CAT基因的表达水平相比,BbcatP基因受H202胁迫的影响最为显著,3h胁迫处理后,其转录水平较正常条件提高13.5倍。结果表明,各CAT在球孢白僵菌生长发育过程中扮演不同的角色,且不同程度地参与氧化胁迫的防御反应。
球孢白僵菌五种过氧化氢酶的生物学功能分析应用根癌农杆菌介导的遗传转化方法成功构建球孢白僵菌各CAT基因的单敲除株。酶活测定显示,正常生长条件下,菌丝CAT活力主要取决于BbcatB、 BbcatP和BbcatD。活性染色PAGE凝胶上仅呈现BbcatB(高)和BbcatP(低)两条酶带,氧化胁迫及热激等胁迫条件并不能改变相应的酶谱类型。抗逆力测定表明,ΔBbcatC和ΔBbcatP菌丝对氧化胁迫十分敏感,50mM H202几乎完全抑制.ΔBbcatP菌落的生长,而其它三种CAT的缺失突变株的菌落生长没有受到明显抑制。ABbcatB菌落生长在含4mM甲萘醌的培养基上明显受抑制,其他CAT基因缺失株生长则差异不显著。在渗透压(NaCl)、杀菌剂(多菌灵)以及热胁迫(33℃)条件下,各敲除株的菌落生长表型均未受到显著影响。另一方面,CAT基因敲除后可显著影响分生孢子对H202、UV-B和高温的耐受力,但影响程度不同。ΔBbcatA、 ΔBbcatP和ΔBbcatC分生孢子对H202的耐受力分别较野生株下降42.6%、57.0%和34.5%,而ΔBbcatB和ΔBbcatD的分生孢子对此氧化胁迫的耐受力未受显著影响。各敲除株的孢子对UV-B辐射的耐受力也表现不同程度的下降,以ΔBbcatB和ΔBbcatD最为显著,较野生株分别下降52.4%和47.6%。耐热力测定显示,ΔBbcatA分生孢子在45℃热胁迫下的半致死时间LT50较野生株下降40%,但其余敲除株都与野生株的LT5o均为52.0min。
毒力测定显示,敲除株ΔBbcatA、 ABbcatP知ΔBbcatD对斜纹夜蛾二龄幼虫的毒力显著低于野生株,其LT50分别较野生株下降1.4天、1.8天和0.9天,而另两个敲除株与野生株无异,其LT5o为5.0～5.1天之间。由此可见,CAT是球孢白僵菌克服昆虫寄主ROS相关的抗氧化酶系,因而是影响菌株重要抗逆性状和毒力的重要因子。
球孢白僵菌内源强启动子Phydl的克隆与优化为了寻找一种可在球孢白僵菌中高表达外源有益基因的内源强启动子,从Bb2860基因组中克隆到I型疏水蛋白基因(hydl)的上游序列,并分别截成-1798(全长、-1290、-1179、-991和-791bp)不同长度的片段或对-1290片段特定转录因子(StuA、 NIT2和Mat-Mc)结合位点施以定点突变后,与绿色荧光蛋白报告基因eGFP融合起来,在球孢白僵菌Bb2860中转化表达,考察各转化子中eGFP蛋白相对于外源通用启动子PgpdA调控的表达水平。结果显示,携带3个必要转录因子的-1290bp为最优的启动子片段(Phydl-t1),由其驱动eGFP表达的转化子菌落的相对荧光强度(RFI)是PgpdA驱动的15.6倍,且eGFP蛋白主要积累于分生孢子形成阶段,尤以成熟的分生孢子中表达量最高。其他截头片段或定点突变的启动子片段启动基因表达的效率均不如Phydl-t1,-791bp片段几乎完全失去启动外源基因表达的能力。在定点突变的片段中,以StuA结合位点的突变影响eGFP基因表达最甚。
高表达中肠特异性杀虫蛋白Vip3Aa1的球孢白僵菌工程株的构建及毒力分析将外源Bt营养期杀虫蛋白基因Vip3Aa1置于优化的内源启动子Phydl-t1下导入野生株Bb2860中,筛选鉴定出一株生长和产孢性状良好且遗传稳定的工程株BbHV8。实时定量PCR和ELISA分析显示,与BbV28菌株(本实验室先前构建由PgpdA启动Vip3Aa1表达的工程菌株)相比,BbHV8培养4天的菌落(菌丝)中Vip3Aa1基因的转录水平上升112倍,在菌丝和培养7天形成的分生孢子中Vip3Aa1蛋白的表达量分别提高7.8和9.8倍。Western杂交和免疫胶体金定位分析表明,Vip3Aa1蛋白在BbHV8分生孢子中高表达且均与分布于细胞质中。孢子被四龄斜纹夜蛾幼虫摄食24h后,能在中肠液中通过Western杂交检测到一条62kDa的活性条带。生测实验表明,BbHV8菌株对斜纹夜蛾二至五龄幼虫均表现出明显的Vip3Aal特有的口服感染毒力,致死虫尸严重萎缩,保湿培养后长出的菌丝稀薄甚至不长。时间-剂量-死亡率(TCM)观察显示,BbHV8的杀虫速度明显快于BbV28和Bb2860,高浓度处理下,BbHV8在处理后第三天能分别杀死100%和53%的二、三龄幼虫,而BbV28和Bb2860在第8天才杀三龄幼虫62%和45%。
TCM模拟分析显示,Bb2860、BbV28和BbHV8对斜纹夜蛾二龄幼虫的LC5o随接种天数差异显著。BbHV8对斜纹夜蛾二龄幼虫的LC5o及其95%置信限由第2天的1293(889～1880)孢子/mm2下将至第5天的26(16～44)孢子/mm2。Bb2860和BbV28对三龄幼虫LC5o的差异不显著,但均显著高于BbHV8的LC5o。基于各菌株的LC5o值,BbHV8处理后3-8天期间对二、三龄幼虫的毒力分别为BbV28的8-80倍和Bb2860的18～931倍,毒力差异倍数随处理后时间延长而缩小。在可比较的处理剂量下,BbHV8对二、三龄幼虫的LTso比BbV28和Bb2860分别缩短1.5～3.8天和2.9～4.6天,即在给定剂量下,BbHV8的杀虫速度比BbV28快63～100%,比Bb2860怏1.1～1.6倍。此外,BbHV8在处理后3-8天杀死90%二至五龄幼虫的孢子剂量为678～1389个孢子/mm2,BbV28和Bb2860对三龄幼虫无可计算的LC5o。以上结果表明,Phydl-t1能强启动外源毒素基因的表达并大量积累毒蛋白于球孢白僵菌分生孢子中,从而大幅提高其对高龄暴食性害虫的毒力。
综上所述,本研究主要成果和创新点在于,一是成功克隆了球孢白僵菌中与甘露醇代谢相关的BbMPD和BbMTD基因,通过基因敲除和表型分析首次揭示了它们在调控该菌生长发育、抗逆及毒力性状中的重要作用。二是首次系统研究了该菌CAT基因种类、结构和功能,阐明了各CAT影响该菌抗逆性状和毒力的程度。三是发现了能驱动外源基因在球孢白僵菌分生孢形成阶段特异性超表达的疏水蛋白启动子Phyd1-t1,并利用其构建了超高表达Vip3Aa1肠毒蛋白的工程菌株,大幅提高了对斜纹夜蛾各龄幼虫的口服毒力,因而是对付暴食性叶面害虫的新武器。这些结果深化了对丝孢类生防真菌抗逆分子机理的认识,形成了真菌杀虫剂遗传改良的关键技术和成果。
As a classic fungal entomopathogen, Beauveria bassiana has been developed into dozens of mycoinsecticides and widely applied in insect pest control. The fungal application is often restricted due to the defect of slow action and the sensitivity to the stresses of high temperature and solar UV irradiation often encountered in summer. Thus, there is an urgent need for exploring possible mechanisms involved in the defence of the fungal pathogen against multiple stresses and improving genetically fungal virulence to target pests.
Mannitol, a small sugar polyol, is widely distributed in fungi and can be served as carbohydrate resources and involved in crucial defense mechanisms that protect cells from damages by environmental stresses. Catalases (CAT) are important antioxidant enzymes in eukaryotic cells and play important roles in protecting cells from the damage of intracellular reactive oxygen species (ROS). However, the regulation of mannitol metabolism and the function of catalases in entomopathogenic fungi are not well understood at present. On the other hand, integrating Bacillus thuringiensis (Bt) vegetative insecticidal protein Vip3Aal into B. bassiana under a constitutive promoter PgpdA from Aspergillus nidulans could increase fungal per os virulence to the neonates of Spodoptera litura but the amount of the target toxin expressed in fungal conidia was too small to kill older larvae. Thus, the present study sought to elucidate a possible mechanism involved in mannitol metabolism and explore the functions of CAT members in B. bassiana in response to multiple stresses by means of gene knockouts and phenotypic analyses. Moreover, a fragment found from the upstream regions of B. bassiana Class I hydrophobin gene (hydl) was used a novel, strong promoter to drive high yield expression of the midgut toxin Vip3Aal in transgenic B. bassiana, yielding an engineered strain highly insecticidal to all instars of S. litura larvae. The results are summarized below.
Characterization of B. bassiana mannitol1-phosphate dehydrogense (BbMPD). A full-length1334-bp fragment of mannitol1-phosphate dehydrogenase gene (BbMPD) was cloned from B. bassiana2860(Bb2860herein), including a putative1176-bp open reading frame (ORF) with a158-bp intron. The gene was found encoding a391amino acid protein and showing56-74%sequence identity to MPDs known from other17fungi. The deduced protein was featured with a putative NADH cofactor binding site (GAGRIG) at the N-terminal. The gene was successfully expressed in E. coli BL21and the recombinant protein (84.4kDa) was well in agreement with the predicted molecular weight. The purified BbMPD showed high substrate specificity to fructose6-phosphote (Fru6P). Its maximal activity in Fru6P reduction was achieved at30℃and pH7.0. In the reaction at optimal 30℃and pH7.0, the BbMPD activities (y) at0.05-5mM Fru6P (x) were well fitted to non-linear Michaelis-Menten equation (r2=0.99), generating the parameters Km and Vmax (±SE) of0.89±0.05mM and913.5114.9U/mg proteins. The catalytic efficiency (kcat/Km) for the reduction of Fru6P by BbMPD was estimated as1.31×104mM-1s-1.
Gene cloning of B. bassiana mannitol dehydrogense (BbMTD). A full-length864-bp fragment of mannitol dehydrogenase gene(BbMTD) was cloned from Bb2860. Three potential stress-responsive elements (AGGGG) and five elements meeting the consensus sequence (SYGGRG) for binding CREA, a broad domain regulator for carbon catabolism repression in Aspergillus, were lcoated in its5'flanking regions. The864-bp ORF with a63-bp intron encoded for a266amino acid protein with the predicted molecular weight of28.2kDa and the isoelectric point of6.6. The deduced protein, featured with a putative NADPH cofactor binding site (GPKGIG) at N-terminal, was found sharing high sequence identity to MTDs known from other fungi.
Expression patterns of BbMPD and BbMTD. The wild-type strain Bb2860was grown on the plates of Saubouraud dextrose agar plus1%yeast extract (SDAY) for7days at25℃to monitor the transcription levels of the two genes in fungal colonies. As a result, the mRNA level of BbMTD increased steadily during the first four-day growth, followed by a20-fold increase on day5relative to day2. Unlike BbMTD, the transcription level of BbMPD fluctuated in a narrow range during the7-day growth and peaked on day4. Under the osmotic stress of1M NaCl) or the oxidative stress of50mM H2O2, the expression levels of both genes increased with stress time. Three-hour stress resulted in an increase of5(NaCl) or11.4(H2O2) fold in BbMTD expression and of10.9(NaCl) and74.9(H2O2) fold in BbMPD expression, respectively. In contrast, both BbMPD and BbMTD expression levels were suppressed by50and63%, respectively, after3-h heat stress at35℃. The results suggest that both genes take important parts in B. bassiana response to multiple stresses.
Functional analyses of BbMPD and BbMTD. Two gene disruption mutants, ABbMPD and ABbMTD, were constructed via Agrobacterium tumefaciens-mediated transformation. Enzymatic activity assays and polyol content analysis showed that both BbMPD and BbMTD were crucial enzymes for mannitol metabolism. The BbMPD disruption resulted in almost all loss of MPD activity to reduce fructose-6-phosphate while the BbMTD knockout led to～80%loss of MTD activity compared with the wild-type strain. The two mutants and Bb2860grown on SDAY plates or incubated in Saubourau dextroe broth (SDB) differed significantly in intracellular mannitol content. The mannitol contents in mycelia and conidia decreased68and83%for△BbMPD, and16and38%for△BbMTD, respectively, compared with the Bb2860counterparts. The mannitol contents of△BbMPD and ΔBbMTD were enhanced respectively by3.0and1.7fold after the SDB-cultured mycelia were transferred to fresh SDB containing1M NaCl for24-h osmotic stress or by40.7and62.3%after exposure to35℃. Accompanied by the decreases of mannitol contents, trehalose contents increased in ΔBbMPD and ΔBbMTD cells under normal or stressed conditions.
The conidial yield of ΔBbMPD on SDAY was significantly reduced by25-27%compared with similar yields of Bb2860and ΔBbMTD. The two mutants showed some differences in colony growth and spore germination on minimal medium with different carbon sources. ΔBbMPD was similar to Bb2860in colony size irrespective of a carbon source but fructose, glucose and mannitol delayed50%spore germination (GT50) for3.0,3.1and6.6h, respectively. In contrast, ΔBbMTD grew much more slowly than Bb2860and its GT50was delayed for7.1h by mannitol but not affected by fructose and glucose. Moreover, ΔBbMPD and ΔBbMTD conidia were38and18%less tolerant to the oxidative stress of H2O2,39and16%less tolerant to the stress of UV-B irradiation, and22and11%less tolerant to the wet-heat stress of45℃, respectively. However, both gene knockout mutants were not affected in conidial tolerance to osmotic stress and and virulence to the apterous adults of green peach aphid Myzus persicae.
Gene cloning, sequence analysis and expression patterns of B. bassiana catalases. Five catalase (CAT) genes were cloned for the first time from B. bassina, including BbcatA, BbcatB, BbcatP, BbcatC and BbcatD. These genes were found encoding three monofunctional catalases (BbcatA, BbcatB, and BbcatC) and two biofunctional catalase-peroxidases (BbcatP and BbcatD). Of those, BbcatC was a small subunit catalase found in B. bassiana and other four were all large subunit catalases. Phylogenetic analysis indicated that five catalases were classified to different groups. BbcatA, a Clade A catalase, is mainly involved in spore-specific processes, such as germination. Its transcription level increased during conidiation but significantly decreased during spore germination and hyphal growth. In SDB cultures including50mM H2O2for3-h oxidative stress, the transcription level of BbcatA was enhanced by4.5fold. BbcatB and BbcatD were Clade B catalases due to the presence of a secretive signal peptide of19amino acids at the N-terminal. The BbcatB gene transcribed increasingly during colony growth, resulting in a peak of30.2fold increase on day7relative to day2. The transcript level of BbcatD was relatively low during the first5-day growth and drastically increased53.1and158.3fold on days6and7, respectively. BbcatC and BbcatP were Clade P members (peroxisomal catalases) due to their peroxisome-targeting signal (PTS). BbcatC transcribed increasingly during colony growth while BbcatP transcibed at a level much lower than other four genes during7-day colony growth. The transcriptional levels of both genes were induced by H2O2added to SDB cultures for3-h oxidative stress, which, for example, increased BbcatP transcription by13.5fold. Our study revealed that the B. bassiana catalases may take different parts in regulating fungal response to oxidative stress.
Functional analyses of five B. bassiana catalases. The disruption mutants of five B. bassiana CAT genes, i.e., ΔBbcatA, ΔBbcatB, ΔBbcatC, ABbcatD and AbbcatP, were constructed by means of A. tumefaciens-mediated transformation. Enzymatic activity assays indicated that BbcatB, BbcatP and BbcatD primarily contributed to the CAT activities of the fungal species under normal conditions. In the profiles of non-denaturing polyacrylamide gels stained with ferricyanide, however, only two CAT-active bands were found in the wild-type strain grown under normal conditions. The upper and lower bands corresponded to BbcatB and BbcatP because they were absent in the ΔBbcatB and ΔbbcatP profiles, respectively.
ΔBbcatC and ΔBbcatP were very sensitive to the oxidative stress of50mM H2O2with their colony growth being greatly suppressed (e.g., nearly100%for AbbcatP). However, other three mutants showed the same response to the oxidative stress as the wild-type strain. All mutant and wild-type strains displayed no significant difference in colony growth on SDAY plates under the stress of1M NaCl,1μg/ml carbendazim or4mM menadione or incubated at33℃. Exceptionally, the colony growth of ABbcatB was suppressed on menadione-inclusive plates. Moreover, conidial tolerance to H2O2was reduced by42.6,57.0and34.5%for ΔBbcatA, ΔBbcatC and AbbcatP, respectively. The conidia of ΔBbcatB and ABbcatD were52.4and47.6%less tolelrance to UV-B irradiation than the wild-type strain, respectively. Conidial tolerance to the heat stress of45℃was similar for all wild-type and mutant strains except AbbcatA, whose median lethal time (LT50) was reduced by40%.
ΔBbcatA, ΔBbcatP and ΔBbcatD displated significantly reduced virulence to the second-instar larvae of S. litura compared to the wild-type strain. Their LT50estimates were delayed for1.4,1.8and0.9days, respectively. The same estimates of ABbcatB, ABbcatC and the wild-type strain fell in the narrow range of5.0-5.1days. Thus, the three catalases are potential virulence factors in B. bassiana.
Great elevation of fungal per os virulence to caterpillars by high yield expression of a midgut-acting toxin in transgenic conidia under a novel promoter. Beuaveria bassiana infects insect pests normally through host cuticle but hardly causes per os infection. We obtained a great progress in high yield expression of Bacillus thuringensis Vip3Aal, a midgut-acting toxin, in transgenic conidia for enhanced fungal virulence to caterpillars through spore ingestion. The toxin production relied on a homologous promoter (Phydl-t1) found to drive the toxin-coding gene expression specifically during conidiation. The gene transcription level in the4-day colonies of all examined transformants was3.4-112fold higher than that in a transgenic strain (BbV28) under the control of PgpdA, a heterologous promoter widely used for gene expression in fungi. The best transformant BbHV8produced9.8-fold-higher Vip3Aal yield in ripe conidia than BbV28. The release of active Vip3Aal into larval midguts from BbHV8conidia ingested within24h was4.4-fold more than that from BbV28. In the bioassays of spore suspensions sprayed onto S. litura larvae (for normal infection) and lotus leaf discs (for feeding), the LC50S of BbHV8against instars II and III on days2-5and3-8were reduced by11-24and8-80fold (declining with time), respectively, compared with the BbV28estimates. BbHV8killed90%instars II-V larvae within3-8days under the economic sprays of678-1389conidia/mm2whereas BbV28and wild-type strain could not kill the older larvae effectively. Conclusively, the high toxin yield in conidia enhanced greatly the toxin release into midguts, the fungal per os virulence to caterpillars and the potential of BbHV8for pest control.
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