胞内海藻糖对球孢白僵菌耐热力的贡献及其代谢相关基因的功能鉴定
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摘要
球孢白僵菌(Beauveria bassiana)是害虫生防真菌的典型代表,其分生孢子制剂的环境稳定性是制约菌剂商品化开发和应用的关键。海藻糖是广泛存在于生物体内的抗逆保护剂,也参与真菌的抗逆境胁迫生理反应,但目前关于生防真菌中海藻糖及其代谢的生物学功能的研究报道有限。为此,本研究提出和验证了“胞内海藻糖参与耐热性”的生物学假设,并通过对海藻糖代谢途径中的中性海藻糖水解酶基因BbNTH1和6-磷酸海藻糖合成酶基因BbTPS1的克隆,将其启动子与报告基因eGFP融合表达和反义RNA干扰表达,研究了它们在多种胁迫下的转录表达情况和抗逆生物学功能。主要研究结果如下所述：
球孢白僵菌胞内海藻糖和甘露醇在热胁迫下的生理响应为了研究胞内海藻糖累积在菌株耐热性中的生理功能,将球孢白僵菌于25℃正常培养3天后进行35、37.5、40℃不同的高温胁迫1-18 h,通过测定胞内海藻糖和海藻糖水解酶活力的变化,以揭示海藻糖代谢在生防真菌中的耐热生理功能。在35、37.5和40℃下胁迫6h后,胞内累积的海藻糖含量平均值从最初的4.2分别迅速上升至88.3、74.7和65.5 mg/g菌丝,但甘露醇的含量却随胁迫温度上升和时间延长而逐渐降低。累积的海藻糖含量与海藻糖水解酶活力(r2=0.73,P<0.01)和甘露醇含量(r2=0.38,P<0.01)显著相关。热胁迫下胞内甘露醇含量与甘露醇脱氢酶(r2=0.41,P<0.01)和1-磷酸甘露醇脱氢酶(r2=0.30,P<0.01)存在显著的负相关。热胁迫后的菌落在25℃下均能恢复生长,但其生长活力取决于热胁迫温度、时间及二者的互作效应(r2=0.98,P<0.01)。将菌落先置于35℃胁迫6h后再在48℃下接受更高温胁迫0-60 min后,其菌落与对照相比表现出更强的耐受力。结果显示,累积的海藻糖部分来源于甘露醇的代谢产物,由此推测甘露醇也可能参与菌株的耐热力。海藻糖水解酶可在热胁迫和恢复过程中降解海藻糖,也可能在菌株耐高温胁迫中起着重要作用。
中性海藻糖水解酶基因克隆、表达纯化及酶学特征分析从球孢白僵菌基因组中克隆到中性海藻糖水解酶基因BbNTH1,其编码蛋白是典型的真菌中性海藻糖水解酶,开放阅读框(ORF)序列长2232 bp,共有三个大小分别为50、49和58 bp的"GT…AG”内含子,编码区翻译后产生一条由743个氨基酸组成的蛋白肽段,预测分子量约为84.4 kDa。在编码蛋白的氨基酸序列中,发现一个依赖于cAMP的磷酸化作用位点(RRGS)和一个钙离子附着位点(DTDGNMQITIED).在大肠杆菌中成功表达了BbNTHl的重组蛋白,其纯化产物的最适反应温度为55℃,最适反应pH 7.0,对海藻糖具有底物专一性,米氏常数Km和Vmax分别为5.05±0.71 mM和1290.98±22.73 U/mg蛋白,降解海藻糖的催化效率(kcat/Km)为2.35x103mM-1s-1。
各种胁迫下调控BbNTHl基因表达的应激响应元件识别为了研究BbNTHl基因的上游调控序列在各种胁迫下的转录表达情况,将其上游启动子序列截短成不同长度的片段(-2713,-1912,-1060和-560 bp),与绿色荧光蛋白报告基因eGFP融合起来,在球孢白僵菌中转化表达,考察eGFP在高温、氧化和高渗透压胁迫下的表达。结果显示,在菌丝和分生孢子形成细胞中eGFP表达最强,但成熟分子孢子中表达较弱。由最短的启动子片段NthP4调控的目标基因在各种胁迫下(40℃热胁迫15-540 min;0.3-1.2 mM甲萘醌氧化胁迫;0.5～1.0 M NaCl渗透压胁迫)的转录表达水平最强。NthP4中存在的两个胁迫响应元件,不论是单个还是两个共同定点突变后,均能导致目标基因在胁迫诱导下的表达下调。这证明在不同胁迫下BbNTHl通过其上游-560 bp启动子序列片段中的两个胁迫响应元件的调控而激活其表达。
6-磷酸海藻糖合成酶基因的克隆及其生物学功能验证从球孢白僵菌基因组中克隆到6-磷酸海藻糖合成酶基因BbTPSl,通过构建含BbTPSl基因RNA干扰发夹结构和筛选标记bar基因的干扰载体在宿主菌株中进行转化表达,经Real Rime PCR筛选出表达量分别下调51.9%和77.5%的反义转化子A2和A4,对其进行了抗逆生物学表型的研究。结果发现,转化子分生孢子在没有添加营养的生理盐水中的萌发率显著低于野生菌株(F2,6=42.5,P<0.01)；转化子孢子耐45℃胁迫的能力显著低于野生菌株(F2,6=29.2,P<0.01)；在35℃胁迫下转化子胞内海藻糖的累积也显著低于野生菌株(FF2,24=3103.9,P<0.01);氧化胁迫(F2,24=17.2,P<0.01)和热胁迫(F2,30=52.7,P<0.01)下转化子菌落活力也显著低于野生菌株。这些结果说明,海藻糖合成酶BbTPS1基因在孢子萌发、孢子耐热力及抗氧化的生理反应中发挥着重要作用。
综上所述,本研究主要成果和创新点,一是研究并建立了热胁迫下菌株胞内海藻糖和甘露醇含量及其相关代谢酶的活力变化的定量评价体系,揭示了球孢白僵菌菌落的耐热力和胞内海藻糖累积的关系。二是克隆了BbNTH1基因,发现其上游启动子最小片段NthP4对其在各种胁迫下的转录表达起关键调控作用,并从中识别出二个关键胁迫响应元件。三是克隆了BbTPSl基因,通过RNA干扰技术抑制其基因表达,证明了其对孢子萌发和耐热力及抗氧化的功能。这些结果有助于深化对丝孢类生防真菌抗逆性状及其生物学背景的认识。
Entomopathogenic fungi are widely applied in microbial control of arthropod pests, being well represented by the fungus Beauveria bassiana. Fungal cells (aerial conidia, blastospores or mycelia) are often formulated as active ingredients of mycoinsecticides and inevitably exposed to the stresses of high temperatures and solar UV irradiation, which are known to affect fungal viability and field performance. Fungal candidate strains with greater stress tolerance are likely to be more potential for successful use in pest control. Fungal trehalose is likely involved in crucial defense mechanisms that protect cells from damages by environmental stresses. However, neither trehalose accumulation nor trehalose metabolism in fungal cells has been related to the tolerance of the fungal biocontrol agents to multiple stresses. Thus, it is necessary to understand possible mechanism of trehalose metabolism in B. bassiana in response to multiple stresses.
This study began from quantitative evaluation changes of intracellular trehalose and mannitol contents and enzymatic activities under thermal stress, resulting in a hypothesis that trehalose accumulation is likely involved in fungal thermotolerance. To confirm the hypothesis, the genes encoding B. bassiana neutral trehalase(BbNTH1) and trehalose-6-phosphate synthase (BbTPS1) were cloned and characterized. Crucial stress-responsive elements (STREs) to control BbNTHl expression under multiple stresses was identified by fusion of the reporter gene eGFP to its promoter. The biological function of BbTPS1 gene was revealed by suppressing its expression using antisense RNA-mediated gene silencing technique. The results are summarized as follows.
Physiological implication of intracellular trehalose accumulation in response of B. bassiana to thermal stress. To explore possible role of intracellular trehalose accumulation in the tolerance of B. bassiana to summer-like thermal stress, three-day colonies of B. bassiana grown on the plates of a glucose-free medium at 25℃were separately exposed to 35,37.5 and 40℃for 1-18 h. Trehalose accumulation in the stressed mycelia increased from initial 4.2 to 88.3,74.7 and 65.5 mg/g biomass after 6-h exposure to 35,37.5 and 40℃respectively while intracellular mannitol level generally declined with more stressful temperatures and longer stress time. The stress-enhanced trehalose level was significantly correlated to the decrease of trehalase activity (r2=0.73) and the level of mannitol (r2=0.38), which was inversely correlated to the activity of mannitol dehydrogenase (r2=0.41) or mannitol 1-phosphate dehydrogenase (r2=0.30) under the stresses. All stressed cultures were successfully recovered at 25℃but their vigor depended on the stress intensity expressed as temperature, time length and the interaction of both (r2=0.98). The highest level of 6-h trehalose accumulation at 35℃was found enhancing the tolerance of the stressed cultures to the greater stress of 48℃for≤60 min. The results suggest that the trehalose accumulation result from metabolized mannitol and contribute to fungal thermotolerance. Trehalase also contributed to the thermotolerance by hydrolyzing accumulated trehalose under the conditions of thermal stress and recovery.
Gene cloning and characterization of neutral trehalase (BbNTHl) from B. bassiana. A full-length 2387-bp fragment of neutral trehalase (BbNTH1) gene was cloned from B. bassiana, including a putative 2232-bp open reading frame with three 50-, 49- and 58-bp putative introns (Genbank accession number:EF122412). In sequence analysis online (http://blast.ncbi.nlm.nih.gov/Blast.cgi), the dediced protein was featured with a cAMP dependent phosphorylation consensus site (RRGS) and a putative calcium binding site (DTDGNMQITIED). The gene was found encoding a 743-aa protein and expressed E. coli BL21 as the recombinant protein of 84.4 kDa in agreement with the predicted molecular weight. The purified BbNTHl showed high substrate specificity to trehalose (100%). Its maximal activity in trehalose hydrolysis was achieved at 55℃and pH 7.0. In the reaction at optimal 55℃and pH 7.0, the BbNTH1 activities (y) at 0.1-1.0 M trehalose (x) were well fitted to the non-linear Michaelis-Menten equation (r2=0.99), generating the parameters Km and Vmax (±SE) of 5.05±0.71 mM and 1290.98±22.73 U/mg protein. The catalytic efficiency (kcat/Km) for the hydrolysis of trehalose by BbNTH1 was estimated as 2.35×103 mM-1 s-1.
Recognition of crucial stress-responsive elements to control BbNTHl expression in response to multiple stresses. To identify crucial stress-responsive elements (STREs) to control BbNTHl expression in response to different stresses, the full-length promoter (-2713 bp) upstream of its open reading frame and three upstream-truncated fragments (-1912,-1060 and -560 bp) were fused to the reporter gene eGFP and then transformed into B. bassiana, respectively. As a result, eGFP was well expressed as intensive fluorescence in mycelia, conidiogenic cells and forming conidia controlled by the full-length promoter with five STREs. Surprisingly, transformant colonies controlled by the shortest fragment with last two STREs at -315 and -274 bp exhibited consistently brightest fluorescence under 7-day oxidative adaption of 0.3-1.2 mM menadione,6-day osmotic stress of 0.5-1 M NaCl and thermal stress of 15-540 min at 40℃after 3-day growth at 25℃. Single or dual site-directed mutations of the two STREs from CCCCT to CATCT significantly altered the gene response to the multiple stresses. Thus, the two STREs in the downstream 560-bp region of the promoter are crucial to regulating not only constitutive but stress-inducible expression of the target gene.
Gene cloning and functional analysis of trehalose-6-phosphate synthase (BbTPSl) from B. bassiana. A trehalose-6-phosphate synthase gene, BbTPS1, was cloned from B. bassiana. The function of BbTPSl was elucidated by suppressing its expression using the method of antisense RNA-mediated gene silencing. Two antisense-RNA plasmids vectoring both the transcriptional element of BbTPS1 hairpin inverted repeat fragments and the gene bar were constructed and then inserted into the genomic DNA of the wild-type strain Bb2860. Expression of BbTPS1 in selected transformants was measured by real time PCR. Transformants A2 and A4 with the suppressed BbTPS1 expression of 51.9% and 77.5% were used in the following experiments to analyze trehalose metabolsin in conidial germination and the role of the gene in response to various stresses. Conidial germination rates of A2 and A4 were significantly lower than that of the wild strain in nutrition-free saline (F2,6=42.5, P<0.01). The silenced expression of BbTPS1 increased significantly the sensitivity of germinating conidia to the 30-min thermal stress of 45℃(F2,6=29.2, P<0.01). The accumulation of trehalose was largely reduced in A2 and A4 mycelia compared to the wild-type strain during thermal stress at 35℃. The colony growths of both transformants and the wild-type strain also showed different responses to the oxidative stress of 1-3 mM menadione (F2,24=17.2, P<0.01) and the thermal stress of 48℃(F2,30=52.7, P<0.01). Our results highlight the important role of BbTPS1 gene in B. bassiana tolerance to the stresses.
In summary, we developed an efficient assay system that enables to accurately quantify changes of intracellular trehalose and mannitol contents and enzymes activities under thermal stress and found that trehalose accumulation takes important part in fungal thermotolerance. The genes encoding B. bassaina neutral trehalase (BbNTH1) and trehalose-6-phosphate synthase(BbTPS1) were characterized for the first time. Two STREs in the downstream 560-bp region of the BbNTH1 promoter were found crucial to regulating not only constitutive but stress-inducible expression of BbNTH1 in response to multiple stresses. Significant contributions of BbTPSl to conidial germination and tolerance to thermal and oxidative stresses were confirmed by means of antisense RNA-mediated gene silencing. These results provide new insights into trehalose involvement in the stress tolerance of B. bassiana and suggest means to improving ecological fitness or field persistence of fungal biocontrol agents.
球孢白僵菌胞内海藻糖和甘露醇在热胁迫下的生理响应为了研究胞内海藻糖累积在菌株耐热性中的生理功能,将球孢白僵菌于25℃正常培养3天后进行35、37.5、40℃不同的高温胁迫1-18 h,通过测定胞内海藻糖和海藻糖水解酶活力的变化,以揭示海藻糖代谢在生防真菌中的耐热生理功能。在35、37.5和40℃下胁迫6h后,胞内累积的海藻糖含量平均值从最初的4.2分别迅速上升至88.3、74.7和65.5 mg/g菌丝,但甘露醇的含量却随胁迫温度上升和时间延长而逐渐降低。累积的海藻糖含量与海藻糖水解酶活力(r2=0.73,P<0.01)和甘露醇含量(r2=0.38,P<0.01)显著相关。热胁迫下胞内甘露醇含量与甘露醇脱氢酶(r2=0.41,P<0.01)和1-磷酸甘露醇脱氢酶(r2=0.30,P<0.01)存在显著的负相关。热胁迫后的菌落在25℃下均能恢复生长,但其生长活力取决于热胁迫温度、时间及二者的互作效应(r2=0.98,P<0.01)。将菌落先置于35℃胁迫6h后再在48℃下接受更高温胁迫0-60 min后,其菌落与对照相比表现出更强的耐受力。结果显示,累积的海藻糖部分来源于甘露醇的代谢产物,由此推测甘露醇也可能参与菌株的耐热力。海藻糖水解酶可在热胁迫和恢复过程中降解海藻糖,也可能在菌株耐高温胁迫中起着重要作用。
中性海藻糖水解酶基因克隆、表达纯化及酶学特征分析从球孢白僵菌基因组中克隆到中性海藻糖水解酶基因BbNTH1,其编码蛋白是典型的真菌中性海藻糖水解酶,开放阅读框(ORF)序列长2232 bp,共有三个大小分别为50、49和58 bp的"GT…AG”内含子,编码区翻译后产生一条由743个氨基酸组成的蛋白肽段,预测分子量约为84.4 kDa。在编码蛋白的氨基酸序列中,发现一个依赖于cAMP的磷酸化作用位点(RRGS)和一个钙离子附着位点(DTDGNMQITIED).在大肠杆菌中成功表达了BbNTHl的重组蛋白,其纯化产物的最适反应温度为55℃,最适反应pH 7.0,对海藻糖具有底物专一性,米氏常数Km和Vmax分别为5.05±0.71 mM和1290.98±22.73 U/mg蛋白,降解海藻糖的催化效率(kcat/Km)为2.35x103mM-1s-1。
各种胁迫下调控BbNTHl基因表达的应激响应元件识别为了研究BbNTHl基因的上游调控序列在各种胁迫下的转录表达情况,将其上游启动子序列截短成不同长度的片段(-2713,-1912,-1060和-560 bp),与绿色荧光蛋白报告基因eGFP融合起来,在球孢白僵菌中转化表达,考察eGFP在高温、氧化和高渗透压胁迫下的表达。结果显示,在菌丝和分生孢子形成细胞中eGFP表达最强,但成熟分子孢子中表达较弱。由最短的启动子片段NthP4调控的目标基因在各种胁迫下(40℃热胁迫15-540 min;0.3-1.2 mM甲萘醌氧化胁迫;0.5～1.0 M NaCl渗透压胁迫)的转录表达水平最强。NthP4中存在的两个胁迫响应元件,不论是单个还是两个共同定点突变后,均能导致目标基因在胁迫诱导下的表达下调。这证明在不同胁迫下BbNTHl通过其上游-560 bp启动子序列片段中的两个胁迫响应元件的调控而激活其表达。
6-磷酸海藻糖合成酶基因的克隆及其生物学功能验证从球孢白僵菌基因组中克隆到6-磷酸海藻糖合成酶基因BbTPSl,通过构建含BbTPSl基因RNA干扰发夹结构和筛选标记bar基因的干扰载体在宿主菌株中进行转化表达,经Real Rime PCR筛选出表达量分别下调51.9%和77.5%的反义转化子A2和A4,对其进行了抗逆生物学表型的研究。结果发现,转化子分生孢子在没有添加营养的生理盐水中的萌发率显著低于野生菌株(F2,6=42.5,P<0.01)；转化子孢子耐45℃胁迫的能力显著低于野生菌株(F2,6=29.2,P<0.01)；在35℃胁迫下转化子胞内海藻糖的累积也显著低于野生菌株(FF2,24=3103.9,P<0.01);氧化胁迫(F2,24=17.2,P<0.01)和热胁迫(F2,30=52.7,P<0.01)下转化子菌落活力也显著低于野生菌株。这些结果说明,海藻糖合成酶BbTPS1基因在孢子萌发、孢子耐热力及抗氧化的生理反应中发挥着重要作用。
综上所述,本研究主要成果和创新点,一是研究并建立了热胁迫下菌株胞内海藻糖和甘露醇含量及其相关代谢酶的活力变化的定量评价体系,揭示了球孢白僵菌菌落的耐热力和胞内海藻糖累积的关系。二是克隆了BbNTH1基因,发现其上游启动子最小片段NthP4对其在各种胁迫下的转录表达起关键调控作用,并从中识别出二个关键胁迫响应元件。三是克隆了BbTPSl基因,通过RNA干扰技术抑制其基因表达,证明了其对孢子萌发和耐热力及抗氧化的功能。这些结果有助于深化对丝孢类生防真菌抗逆性状及其生物学背景的认识。
Entomopathogenic fungi are widely applied in microbial control of arthropod pests, being well represented by the fungus Beauveria bassiana. Fungal cells (aerial conidia, blastospores or mycelia) are often formulated as active ingredients of mycoinsecticides and inevitably exposed to the stresses of high temperatures and solar UV irradiation, which are known to affect fungal viability and field performance. Fungal candidate strains with greater stress tolerance are likely to be more potential for successful use in pest control. Fungal trehalose is likely involved in crucial defense mechanisms that protect cells from damages by environmental stresses. However, neither trehalose accumulation nor trehalose metabolism in fungal cells has been related to the tolerance of the fungal biocontrol agents to multiple stresses. Thus, it is necessary to understand possible mechanism of trehalose metabolism in B. bassiana in response to multiple stresses.
This study began from quantitative evaluation changes of intracellular trehalose and mannitol contents and enzymatic activities under thermal stress, resulting in a hypothesis that trehalose accumulation is likely involved in fungal thermotolerance. To confirm the hypothesis, the genes encoding B. bassiana neutral trehalase(BbNTH1) and trehalose-6-phosphate synthase (BbTPS1) were cloned and characterized. Crucial stress-responsive elements (STREs) to control BbNTHl expression under multiple stresses was identified by fusion of the reporter gene eGFP to its promoter. The biological function of BbTPS1 gene was revealed by suppressing its expression using antisense RNA-mediated gene silencing technique. The results are summarized as follows.
Physiological implication of intracellular trehalose accumulation in response of B. bassiana to thermal stress. To explore possible role of intracellular trehalose accumulation in the tolerance of B. bassiana to summer-like thermal stress, three-day colonies of B. bassiana grown on the plates of a glucose-free medium at 25℃were separately exposed to 35,37.5 and 40℃for 1-18 h. Trehalose accumulation in the stressed mycelia increased from initial 4.2 to 88.3,74.7 and 65.5 mg/g biomass after 6-h exposure to 35,37.5 and 40℃respectively while intracellular mannitol level generally declined with more stressful temperatures and longer stress time. The stress-enhanced trehalose level was significantly correlated to the decrease of trehalase activity (r2=0.73) and the level of mannitol (r2=0.38), which was inversely correlated to the activity of mannitol dehydrogenase (r2=0.41) or mannitol 1-phosphate dehydrogenase (r2=0.30) under the stresses. All stressed cultures were successfully recovered at 25℃but their vigor depended on the stress intensity expressed as temperature, time length and the interaction of both (r2=0.98). The highest level of 6-h trehalose accumulation at 35℃was found enhancing the tolerance of the stressed cultures to the greater stress of 48℃for≤60 min. The results suggest that the trehalose accumulation result from metabolized mannitol and contribute to fungal thermotolerance. Trehalase also contributed to the thermotolerance by hydrolyzing accumulated trehalose under the conditions of thermal stress and recovery.
Gene cloning and characterization of neutral trehalase (BbNTHl) from B. bassiana. A full-length 2387-bp fragment of neutral trehalase (BbNTH1) gene was cloned from B. bassiana, including a putative 2232-bp open reading frame with three 50-, 49- and 58-bp putative introns (Genbank accession number:EF122412). In sequence analysis online (http://blast.ncbi.nlm.nih.gov/Blast.cgi), the dediced protein was featured with a cAMP dependent phosphorylation consensus site (RRGS) and a putative calcium binding site (DTDGNMQITIED). The gene was found encoding a 743-aa protein and expressed E. coli BL21 as the recombinant protein of 84.4 kDa in agreement with the predicted molecular weight. The purified BbNTHl showed high substrate specificity to trehalose (100%). Its maximal activity in trehalose hydrolysis was achieved at 55℃and pH 7.0. In the reaction at optimal 55℃and pH 7.0, the BbNTH1 activities (y) at 0.1-1.0 M trehalose (x) were well fitted to the non-linear Michaelis-Menten equation (r2=0.99), generating the parameters Km and Vmax (±SE) of 5.05±0.71 mM and 1290.98±22.73 U/mg protein. The catalytic efficiency (kcat/Km) for the hydrolysis of trehalose by BbNTH1 was estimated as 2.35×103 mM-1 s-1.
Recognition of crucial stress-responsive elements to control BbNTHl expression in response to multiple stresses. To identify crucial stress-responsive elements (STREs) to control BbNTHl expression in response to different stresses, the full-length promoter (-2713 bp) upstream of its open reading frame and three upstream-truncated fragments (-1912,-1060 and -560 bp) were fused to the reporter gene eGFP and then transformed into B. bassiana, respectively. As a result, eGFP was well expressed as intensive fluorescence in mycelia, conidiogenic cells and forming conidia controlled by the full-length promoter with five STREs. Surprisingly, transformant colonies controlled by the shortest fragment with last two STREs at -315 and -274 bp exhibited consistently brightest fluorescence under 7-day oxidative adaption of 0.3-1.2 mM menadione,6-day osmotic stress of 0.5-1 M NaCl and thermal stress of 15-540 min at 40℃after 3-day growth at 25℃. Single or dual site-directed mutations of the two STREs from CCCCT to CATCT significantly altered the gene response to the multiple stresses. Thus, the two STREs in the downstream 560-bp region of the promoter are crucial to regulating not only constitutive but stress-inducible expression of the target gene.
Gene cloning and functional analysis of trehalose-6-phosphate synthase (BbTPSl) from B. bassiana. A trehalose-6-phosphate synthase gene, BbTPS1, was cloned from B. bassiana. The function of BbTPSl was elucidated by suppressing its expression using the method of antisense RNA-mediated gene silencing. Two antisense-RNA plasmids vectoring both the transcriptional element of BbTPS1 hairpin inverted repeat fragments and the gene bar were constructed and then inserted into the genomic DNA of the wild-type strain Bb2860. Expression of BbTPS1 in selected transformants was measured by real time PCR. Transformants A2 and A4 with the suppressed BbTPS1 expression of 51.9% and 77.5% were used in the following experiments to analyze trehalose metabolsin in conidial germination and the role of the gene in response to various stresses. Conidial germination rates of A2 and A4 were significantly lower than that of the wild strain in nutrition-free saline (F2,6=42.5, P<0.01). The silenced expression of BbTPS1 increased significantly the sensitivity of germinating conidia to the 30-min thermal stress of 45℃(F2,6=29.2, P<0.01). The accumulation of trehalose was largely reduced in A2 and A4 mycelia compared to the wild-type strain during thermal stress at 35℃. The colony growths of both transformants and the wild-type strain also showed different responses to the oxidative stress of 1-3 mM menadione (F2,24=17.2, P<0.01) and the thermal stress of 48℃(F2,30=52.7, P<0.01). Our results highlight the important role of BbTPS1 gene in B. bassiana tolerance to the stresses.
In summary, we developed an efficient assay system that enables to accurately quantify changes of intracellular trehalose and mannitol contents and enzymes activities under thermal stress and found that trehalose accumulation takes important part in fungal thermotolerance. The genes encoding B. bassaina neutral trehalase (BbNTH1) and trehalose-6-phosphate synthase(BbTPS1) were characterized for the first time. Two STREs in the downstream 560-bp region of the BbNTH1 promoter were found crucial to regulating not only constitutive but stress-inducible expression of BbNTH1 in response to multiple stresses. Significant contributions of BbTPSl to conidial germination and tolerance to thermal and oxidative stresses were confirmed by means of antisense RNA-mediated gene silencing. These results provide new insights into trehalose involvement in the stress tolerance of B. bassiana and suggest means to improving ecological fitness or field persistence of fungal biocontrol agents.
引文
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