【作者】 周刚；

【导师】 冯明光；

【作者基本信息】 浙江大学 ， 微生物学， 2011， 博士

【摘要】 基于绿僵菌的真菌杀虫剂被广泛用于如蝗虫等重要农业害虫的防治。作为有效成份为分生孢子或菌丝侵染体的绿僵菌制剂,在田间应用中常遭受高温、阳光紫外辐射、化学杀菌剂施用等环境和人为胁迫因素的影响,使侵染体活力降低乃至失活而影响真菌制剂杀虫效果的稳定性。基于此,本研究以30株野生绿僵菌对苯并咪唑类杀菌剂一一多菌灵抗性的评价为起点,通过微管蛋白的化学诱变和定点突变,探讨金龟子绿僵菌多菌灵抗性的成因；通过分别构建金龟子绿僵菌近缘种一一罗伯茨绿僵组氨酸激酶和腺苷酸环化酶的基因敲除株、回补株并进行一系列表型及其关联基因转录水平的分析,揭示了两种酶调控该菌生长发育、产孢、不同抗逆性状及毒力的生物学功能。所获结果摘要如下：绿僵菌的多菌灵抗性及其协变抗逆性状分析对寄主和地理来源不同的21株金龟子绿僵菌Metarhizium anisopliae.8株蝗绿僵菌M. acridum和1株金龟子绿僵菌大孢变种M. anisopliae var. majus的多菌灵抗性水平测定的结果,仅1株金龟子绿僵菌(Man456)表现低抗,抑制99%菌落形成的最低浓度MIC为5.76μg/mL,而其余29株对多菌灵都表现为敏感,平均MIC仅为2.47(1.14～4.98)μg/mL。以Man456为出发菌株进行亚硝酸钠的化学诱变并在提高的多菌灵胁迫下进行选择,获得11株高抗多菌灵的突变株,MIC均大大超过1000μg/mL,其中5株的ECso(抑制50%菌落形成的多菌灵浓度)也超过1000μg/mL,另6个突变株的EC50介于24～447μg/mL。伴随着多菌灵抗性的大幅提高,所有突变株分生孢子对48℃湿热胁迫的耐受力均大幅或显著下降,且对UV-B辐射的耐受力也发生显著变化。从出发菌株及其11个高抗诱变株中克隆出各自的α-微管蛋白和β-微管蛋白基因序列,经比对分析,发现在α-微管蛋白和p-微管蛋白的序列中分别存在共同的氨基酸残基点突变S231L和S144G。采用基因重叠延伸(SOE)法,对野生菌株Man456实施两种微管蛋白公共敏感位点的定点突变,所获定点突变株aT-S231L和βT-S144G对多菌灵的抗性也显著提高,而且孢子耐受热胁迫和UV-B辐射的能力也发生相应变化。然而,两种微管蛋白公共敏感位点的定点突变后多菌灵抗性的变化,不足以解释大多数化学诱变株对多菌灵的超常高抗性,即绿僵菌对多菌灵的高抗性可能存在微管蛋白突变以外的机制。组氨酸激酶调节绿僵菌系列生物学性状的功能分析组氨酸激酶histidine kinase(HK)位于高渗甘油代谢途径(HOG pathway)的上游,一般认为会参与真菌抗逆性状及生长发育的调控。罗伯茨绿僵菌(M. robertsii)的组氨酸激酶(mhkl)与已知的99种真菌HK的序列同源性为74-100%,属于Group III HK。运用农杆菌介导法对mhkl基因成功地实施敲除和回补,用所获敲除株Δmhkl与回补株Δmhk1/mhk1及野生株进行一系列表型变化的实验和分析,发现mhkl敲除后的主要表型变化包括：(1)敲除株在常规培养条件下的产孢量提高50～67%；(2)菌落生长对NaC1、KC1、葡萄糖和蔗糖的高渗透压胁迫的耐受力提高13-46%；(3)菌落生长对甲萘醌氧化胁迫表现为敏感性升高,而对H202氧化胁迫表现为耐受力增强；(4)分生孢子对48℃湿热胁迫的耐受力提高27-37%；(5)分生孢子中甲酸可抽提的孢壁蛋白含量提高41～51%；(6)分生孢子对UV-B辐射的耐受力下降约6%；(7)分生孢子对黄粉虫三龄幼虫的侵染力下降约25%。实时定量PCR(qRT-PCR)检测各菌株中18个表型相关基因的转录水平,发现敲除株中HOG途径中的3个关键蛋白激酶基因mapkkk、 mapkk及mapk的表达上调3.7～8.5倍,2个产孢相关基因flbC和hymA的表达分别上调4.7和1.6倍,3种过氧化氢酶(cAT)基因的表达上调1.6～15.1倍,而3种超氧化物歧化酶(SOD)的表达则下调67～89%,而其余的CAT和SOD基因表达则无显著变化。这些结果显示,mhkl在罗伯茨绿僵菌产孢、多胁迫响应及侵染力的调控中起着重要作用,但不参与二甲酰亚胺类杀菌剂一一菌核净的抗性调控。腺苷酸环化酶调节绿僵菌生长与抗逆性状的功能分析真菌的腺苷酸环化酶adenylate cyclase (ACY)能将ATP催化成cAMP,使生成的cAMP诱导cAMP依赖型蛋白激酶protein kinase (PKA)的表达,因而是细胞cAMP-PKA信号传导途径中的关键酶。罗伯茨绿僵菌ACY(记为macy)与已知的98种真菌ACY的序列同源性介于23～94%之间。用农杆菌介导法对macy基因予以敲除和回补,同回补株Δmacy/macy及野生株相比,敲除株Δmacy发生了一系列显著的表型变化：(1)在正常条件下的菌落生长速率和产孢量大幅下降；(2)盐渗透压胁迫对其菌落生长的抑制作用小于野生株和回补株,而糖渗透压胁迫能促进其菌落生长；(3)10mM甲萘醌氧化胁迫完全抑制菌落生长,而100mM H2O2氧化胁迫大幅促进其菌落生长；(4)cu2+、Mg2+、 Mn2+和Ca2+等金属离子对其菌落生长表现为不同程度的抑制作用,而Fe3+表现为促进菌落生长；(5)在查氏平板上,菌落生长对碳源的利用,以蔗糖最佳,以半乳糖最差；而NH4Cl和NaNO2都能大大促进菌落生长；(6)分生孢子的耐热力、甲酸可抽提孢壁蛋白含量和耐UV-B紫外辐射的能力均下降；(7)分生孢子对黄粉虫三龄幼虫的侵染力下降22～29%。例外地,菌核净对各菌株的抑制作用仅在高浓度下略有差异。用qRT-PCR方法检测各菌株中17个表型相关基因的转录水平,发现敲除株cAMP-PKA信号通路中的pka基因的表达量上调3.93倍,微循环产孢基因mmc的表达下调82%,cat1和cat3的表达分别上调4.2和11.6倍,sod4、 sod、和sod6的表达下调87～89%,其余受检的产孢相关基因、CAT基因和SOD基因的表达则无显著变化。这些结果表明,罗伯茨绿僵菌的腺苷酸环化酶参与生长发育、多胁迫响应及侵染力等重要生物学性状的调控,但同样与菌核净的抗性调控无关。更多还原

【Abstract】 Metarhizium-based mycoinsecticides have been widely applied in the control of insect pests, such as locusts and grasshoppers. As the active ingredients of fungal formulations, formulated fungal cells, such as conidia, are often exposed to environmental stresses including high temperatures, solar UV irradiation and fungicides applied for plant disease control in the field, suffering from some degree of viability loss that affects the field persistence and efficacy of a fungal application. This study started from evaluating the resistance of30wild-type Metarhizium strains to carbendazim (benzimidazole fungicide), followed by examining common site mutations in the tubulins of carbendazim-resistant mutants from chemical mutagenesis in order to probe the cause of the resistance, and constructing the knockout and complement mutants of Metarhizium robertsii histidine kinase (mhkl) and adenylate cyclase (macy) for various phenotypic analyses in order to reveal the roles of the two enzymes in regulating growth, development, sproulation, multi-stress responses and infectivity, respectively. The results are summarized below.Metarhizium resistance to carbendazim and associated changes in response to multiple stresses. Thirty wild-type strains consisting of21Metarhizium anisopliae, eight M. acridum and one M. anisopliae var. majus with different host and geographic origins were assayed for their sensitivities to carbendazim (methyl2-benzimidazole carbamate, MBC herein) using a gradient-concentration system. Of those, only one M. anisopliae strain (Man456) showed low resistance due to the minimal inhibition concentration (MIC) of5.76μg/ml for99%inhibition of colony formation while all other29strains were sensitive to the fungicide with the mean MIC of2.47(1.14-4.98)μg/ml. The selected strain, Man456, was subjected to NaNO2-induced mutagenesis, generating11mutants highly resistant to MBC. The MICs of all mutants exceeded1000μg/ml. Five of mutants even had the EC50(an MBC concentration suppressing50%colony fomation) of>1000μg/ml and the rest mutants had the EC50from24to447μg/ml. Accompanied by the enhanced MBC resistance, conidial tolerance to wet-heat stress at48℃was reduced for all the mutants and their UV-B resistance also changed. Two genes encoding a-tubulin and (3-tubulin were cloned separately from the wild-type strain and the MBC-resistant mutants by means of the technique of splicing-by-overlap extension (SOE), followed by online analysis of their protein sequences. As a result, the common site mutations S231L and S144G were found in the a-tubulin and β-tubulin sequences of all mutants, respectively. The same site-directed mutations were constructed in the wild-type strain, yielding mutants αT-S231L and βT-S144G. Both mutants showed higher resistance to MBC than wild-type, lower tolerance to the wet-heat stress and altered UV-B resistance. However, the changes in the MBC resistance of the two site-directed mutants are not enough to elucidate the extraordinarily high MBC resistance in the mutants from chemical mutagenesis. This suggests other mechanism(s) likely involved in the high MBC resistance of M. anisopliae.Characterized functions of M. roberstii histidine kinase (mhkl). The role of M. robertsii Group III histidine kinase(mhkl) in regulating various phenotypes of the fungal entomopathogen and the transcripts of the genes encoding three mitogen-activated protein (MAP) kinases in HOG-pathway and13proteins possibly associated with the phenotypes were probed by constructing Amhkl and Amhkl/mhkl mutants via Agrobacterium-mediated transformation. All examined Amhkl phenotypes except no change in fungicide (dimethachlon) sensitivity differed significantly from those of wild-type and Amhkl/mhkl, which were similar or close to each other in all phenotypic parameters. Significant main phenotypic changes in Amhkl included50-67%increases in conidial yield on two normal media,13-46%increases in osmotolerance to salts and sugars,27-37%increases in conidial thermotolerance at48℃,41-51%increases in formic-acid-extractable protein content relating to the thermotolerance,～6%reduction in conidial UV-B resistance, and-25%decrease in virulence to Tenebrio molitor larvae. Quantitative real-time RT-PCR (qRT-PCR) indicated that the mhkl disruption caused3.7-8.5-fold up-regulation of three MAP kinase genes(mapkkk, mapkk and mapk),1.6-4.7-fold up-regulation of two conidiation-related genes (flbC and hymA),1.6-15.1-fold up-regulation of three catalase (CAT) genes, and67-89%down-regulation of three superoxide dismultase (SOD) genes, but little change in the transcriptional expression of other CAT and SOD genes examined. The results provide new insights into the crucial role of mhkl in regulating the downstream genes and associated phenotypes important for the fungal biocontrol potential.Characterized functions of M. roberstii adenylate cyclase. Fugnal adenylate cyclase (ACY) enables to catalyze ATP into cAMP to activate cAMP-dependent protein kinase (PKA), thereby acting as a crucial enzyme in the pathway of cAMP-PKA signaling transduction. The sequence of M. roberstii ACY (macy) showed23-94%identity to98ACY sequences of other fungi. The macy gene was successfully disrupted and complemented via Agrobacterium-mediated transformation. Compared to Δmacy/macy, and WT, the disruptant Δmacy had significant phenotypic changes. These included:(1) reduced colony growth and greatly decreased sporulation capacity under normal conditions;(2) colony growth less suppressed by the osmotic stress of KC1or NaCl but facilitated by the osmotic stress of glucose or sucrose;(3) colony growth inhibited by10mM menadione but greatly facilitated by100mM H2O2;(4) colony growth facilitated by Fe3+but suppressed by Cu2+, Mg2+, Mn2+and Ca2+in medium;(5) colony growth less sppressed by sucrose than galactose as a mere carbon source but greatly facilitated by NH4CI or NaNO2as a mere nitrogen source;(6) significant dicreases in conidial thermotolerance, formic-acid-extractable conidial protein content and conidial UV-B tolerance;(7)22-29%reduction in conidial infectivity to T. molitor larvae. Exceptionally, the Amacy sensitivity to dimethachlon had little change at the concentration of200μg/ml. The qRT-PCR analyses indicated that the macy knockout resulted in3.93fold up-regulation of pka gene in the cAMP-PKA pathway,82%down-regulation of a gene (mmc) associated with microcycle conidiation,4.2and11.6fold up-regulation of two CAT genes (catl and cat3), and87-89%down-regulation of three SOD genes (sod4, sod5and sod6) but little change in the transcript levels of other10genes, including flbC, hymA, five CAT genes and three SOD genes. The results provided the first overview to the crucial role of macy in regulating the growth, development, sporulation, multi-stress responses and infectivity of M. robertsii.更多还原