球孢白僵菌甘露糖转移酶、组蛋白乙酰转移酶、叉头转录因子及6-磷酸海藻糖合成酶的功能解析及其同生物防治潜能的关联

发布时间：2018-09-15 05:28

【摘要】：球孢白僵菌(Beauveria bassiana)是广泛用于农林害虫生物防治的昆虫病原真菌,其侵染体是分生孢子,也是许多真菌杀虫剂的活性成分。分生孢子制剂防治田间害虫的效果往往受到温度、湿度、紫外辐射及化学农药等多种环境胁迫的影响。因此,探索生防真菌抗逆境胁迫的生理机制,对以提高其生防潜能为目标的菌株遗传改良及菌剂的合理应用具有重要意义。本研究围绕这一目标,一是解析了球孢白僵菌甘露糖转移酶PMT和Ktr家族的功能,揭示了它们对细胞生长发育、环境抗逆性及寄主侵染力的重要贡献。二是研究解析了乙酰转移酶Mst2对球孢白僵菌生长发育、胁迫应答、细胞周期和毒力的影响。三是解析forkhead转录因子Fkh2的功能,发现Fkh2通过调节相关基因的转录而参与球孢白僵菌细胞周期、无性发育及毒力的调控。四是解析了球孢白僵菌中两个海藻糖合成酶同源蛋白TpsA和TpsB对海藻糖合成、生长发育、孢子质量、多胁迫响应及毒力的贡献,发现二者对几乎所有生防潜能相关性状的贡献都表现为加性效应。主要的研究内容和结果概述如下：球孢白僵菌PMT家族甘露糖转移酶的功能解析PMT家族是在内质网上催化第一个甘露糖转移到蛋白质丝氨酸/苏氨酸上的一类甘露糖转移酶,根据结构特征可分为PMT1、PMT2和PMT4三个亚族。这三个亚族都存在于球孢白僵菌中且每个亚族只有一个成员,分别为Pmtl、Pmt2和Pmt4。其中Pmt2是敲除致死的,通过构建Pmt2的RNA干扰菌株和Pmtl及Pmt4的单基因敲除/回补菌株并进行多种表型分析,三个基因的功能得到较为详尽的解析。与野生型相比,单基因缺失或RNA干扰菌株在生长、产孢、孢子活力、多胁迫耐受力及毒力等诸方面均表现不同程度的缺陷。其中,Pmt2的三个干扰菌株、APmtl及ΔPmt4在富营养和限制培养基上的生长减慢20～79%,分生孢子产量下降16～72%,并伴随孢子活力显著下降即萌发一半所需的时间显著延长。不仅如此,干扰及敲除菌株在菌丝生长或孢子萌发期间,对氧化、高渗、胞壁干扰、高温及UV-B辐射等环境胁迫的抵抗力都有不同程度的显著下降。在对大蜡螟(Galleria mellonella)幼虫的生物测定中,突变株经正常体壁侵染的毒力下降53～62%,但通过血腔注射的毒力未发生大的变化。值得一提的是,突变株的孢壁完整性遭受很大程度的破坏,包括孢壁变薄、孢子表面疏水性降低及重要孢壁成分发生改变。所有表型的变化都在回补株中得到恢复。因此,PMT家族在球孢白僵菌中都不是功能冗余的,而是各成员相互协调共同调节宿主菌对复杂多样环境的适应性及对昆虫寄主的侵染力,因而在球孢白僵菌生防潜能的维持中发挥着互不替代的重要作用。球孢白僵菌Ktr家族甘露糖转移酶的功能比较Ktr家族甘露糖转移酶的功能是将第二个甘露糖连接到被PMT家族成员转移的第一个甘露糖基上,因而是对糖蛋白甘露糖链进行延伸的转移酶,其调控的反应发生在高尔基体内。球孢白僵菌有三个Ktr家族成员,分别为Ktrl、Ktr4和Kre2/Mnt1。通过单基因敲除/回补菌株的构建并进行表型分析,发现Δktr4和Δkre2的分生孢子产量大幅下降～92%,孢子活力显著降低,孢子大小和复杂度也发生显著变化；两个敲除菌株对培养基中不同碳、氮源营养的摄入利用率不如野生株,表现为菌落生长缓慢。与野生型相比,Δktr1在孢子产量及菌落生长方面并无显著变化,但细胞壁中甘露糖蛋白和几丁质等成分的变化都远大于Δktr4和Δkre2,菌丝细胞壁变薄,孢子细胞壁表面疏水性下降12%。Δktr4和Δkre2细胞壁中α-葡聚糖含量显著高于野生型和Δktrl,孢子壁厚度也比Δktr1更薄,孢子表面疏水性分别下降64%和71%。总体上,Δktr4和Δkre2比Aktrl对氧化和胞壁干扰胁迫更敏感,但三者对高渗胁迫的反应则差异不大。高温、UV-B辐射耐受性以及毒力在Δktr4和Δkre2中都大大下降,而Δktrl只对高温敏感。结果显示,Ktr1、Ktr4和Kre2对球孢白僵菌生防潜能都有不同程度的贡献,但Ktr4和Kre2的贡献远大于Ktr1的贡献。球孢白僵菌组蛋白乙酰转移酶Mst2的功能解析组蛋白中赖氨酸的乙酰化与基因的转录活性密切相关,但由于多数乙酰转移酶和去乙酰转移酶对赖氨酸的特异性修饰作用知之有限,每个赖氨酸乙酰化与细胞功能之间的关系目前并不十分清楚。球孢白僵菌中有一个组蛋白乙酰转移酶Mst2,它与裂殖酵母(Schizosaccharomyces pombe)乙酰转移酶SpMst2同源,并能特异性乙酰化组蛋白3(H3)的第14位赖氨酸(H3K14)。为了明确Mst2在球孢白僵菌中的作用,我们构建和分析了Mst2的敲除／回补菌株。表型实验显示,AMst2对不同碳氮源的利用能力远不如野生株和回补株,正常培养条件下的产孢能力受损较大,孢子活力下降,抵抗氧化、高渗及胞壁干扰胁迫的能力显著下降。敲除株分生孢子耐高温、抗紫外能力显著减弱,表面疏水性和对敏感昆虫的侵染力均下降。细胞流式分析显示,AMst2的芽生孢子变小,密度降低,细胞周期中S期延长,G2/M期缩短。综合所有分析结果,Mst2在DNA损伤节点上起着关键作用,因而参与调控球孢白僵菌的细胞周期、无性发育、多胁迫应答及其对昆虫寄主的侵染力。球孢白僵菌叉头转录因子Fkh2的功能解析模式丝状真菌一般拥有叉头Forkhead (FKH)转录因子Fkhl和Fkh2,主要参与细胞周期的调控而影响生物学性状。不同于模式丝状真菌,球孢白僵菌只有Fkh2,不存在Fkhl。将Fkh2编码基因从野生株中敲除,导致球孢白僵菌细胞周期发生紊乱,在不同碳氮源培养基上的菌落生长不同程度地减慢,而且敲除菌株的菌丝隔膜增多,菌丝细胞粗短。有趣的是,敲除株的产孢提前,在正常平板培养条件下分生孢子产量显著升高,在液培条件下芽生孢子的产量也显著高于野生株,但两种孢子都明显变小,且密度降低,显示孢子内含物减少。其分生孢子对氧化和高渗胁迫的敏感性升高,耐UV-B辐射和高温的能力减弱,说明Fkh2可能参与宿主菌的胁迫应答。此外,用分生孢子悬液对大蜡螟幼虫进行体壁侵染和注射侵染的生物测定,结果显示敲除菌株的毒力显著降低。敲除菌株所有这些表型变化都在若干表型相关功能基因的转录分析中获得支持,并且都在回补菌株中得到很好的恢复。结果显示,Fkh2不仅参与调控球孢白僵菌的细胞周期循环,而且还调控生长发育、多胁迫应答及毒力等多种生防潜能相关的性状。球孢白僵菌中两种6-磷酸海藻糖合成酶同源物的功能解析海藻糖的生物合成途径对于动植物病原真菌是非常重要的,因为细胞内海藻糖的积累水平关系到宿主菌的环境适应性和寄主侵染力。球孢白僵菌有两个6-磷酸海藻糖合成酶(TPS),分别为TpsA和TpsB。通过单基因、双基因敲除菌株及回补菌株的构建与分析,发现双敲菌株△tpsA△tpsB的菌丝细胞中既检测不到TPS的酶活,也检测不到任何海藻糖的积累；而单敲菌株△tpsA的TPS酶活和海藻糖积累水平在正常和同胁迫条件下分别下降71～75%和72～80%,在△tpsB中分别下降21～30%和15～45%。两个单敲株在给定条件下TPS酶活损失或海藻糖积累水平下降幅度之和,正好接近双敲菌株的酶活损失或海藻糖含量的下降幅度。正常培养条件下分生孢子产量在双敲株中下降达98%,而在△tpsA和△tpsB中分别下移33%和50%。有趣的是,表征孢子质量的海藻糖含量、孢壁结构成分、疏水性、活力、大小及密度均在双敲株中受损最严重,后依次为△tpsA和△tpsB。相同的趋势也见于三个敲除株对氧化、高渗、胞壁干扰、高温及UV-B紫外辐射等环境胁迫抵抗力的缺陷变化以及对大蜡螟幼虫毒力的缺陷变化。这些结果证明,球孢白僵菌中TpsA对海藻糖合成、营养生长、孢子质量、多胁迫应答及寄主侵染过程的调控作用均大于TpsB,后者仅表现比前者稍强的产孢调控作用。最重要的是,TpsA和TpsB对球孢白僵菌每种表型的调控作用都表现为加性效应,不同于一些模式丝状真菌中多个TPS同源物不一定都起作用的研究报道。因此,两个TPS同源蛋白对球孢白僵菌适应不同类型昆虫寄主及其复杂多样环境的生存方式具有特别重要的意义。
[Abstract]:Beauveria bassiana is an insect pathogenic fungus widely used in biological control of agricultural and forestry pests. Its infectious body is conidia, and it is also an active component of many fungal insecticides. Therefore, it is of great significance to explore the physiological mechanism of biocontrol fungi against stress in order to improve the genetic improvement of strains and the rational application of fungicides. Secondly, the effects of acetyltransferase Mst2 on the growth and development, stress response, cell cycle and virulence of Beauveria bassiana were studied. Thirdly, the function of forkhead transcription factor Fkh2 was analyzed. It was found that Fkh2 participated in the cell cycle, asexual development and virulence of Beauveria bassiana by regulating the transcription of related genes. Fourthly, the contribution of two trehalose synthase homologues, TpsA and TpsB, to trehalose synthesis, growth and development, spore quality, multiple stress response and virulence of Beauveria bassiana was analyzed. It was found that the contribution of TpsA and TpsB to almost all traits related to biocontrol potential was additive. Below: Functional analysis of mannose transferases in the PMT family of Beauveria bassiana The PMT family is a class of mannose transferases catalyzed by the first mannose transfer from the endoplasmic reticulum to the protein serine/threonine. According to their structural characteristics, they can be divided into three subgroups: PMT1, PMT2 and PMMT4. There was only one member, Pmtl, Pmt2 and Pmt4. Pmt2 was knockout lethal. The function of the three genes was analyzed in detail by constructing RNA-interfering strains of Pmt2, single gene knockout/replenishment strains of Pmtl and Pmt4, and various phenotypes were analyzed. The growth of APmtl and_Pmt4, three interfering strains of Pmt2, slowed down by 20-79% in eutrophic and restricted medium, and the conidia yield decreased by 16-72%, and the time required for germination was prolonged significantly with the decrease of spore activity. In addition, the resistance to oxidative stress, hyperosmotic stress, cell wall disturbance, high temperature and UV-B radiation decreased significantly during mycelial growth or spore germination. It is worth mentioning that the integrity of the spore wall of the mutant strain was damaged to a great extent, including the thinning of the spore wall, the decrease of the hydrophobicity of the spore surface and the change of the important spore wall components. All phenotypic changes were restored in the restored strain. Therefore, the PMT family was not found in Beauveria bassiana. Functional redundancy means that each member coordinates with each other to regulate the adaptability of host bacteria to complex and diverse environments and their infectivity to insect hosts, thus playing an irreplaceable role in maintaining the biological control potential of Beauveria bassiana. Functional comparison of mannose transferases of the Ktr family of Beauveria bassiana with those of the Ktr family The function is to attach the second mannose to the first mannose group transferred by a member of the PMT family and thus to extend the glycoprotein mannose chain. The regulatory response occurs in the Golgi apparatus. Beauveria bassiana has three Ktr family members, namely, Ktrl, Ktr4 and Kre2/Mnt1. Phenotypic analysis showed that the conidia yield of ktr4 and kre2 decreased significantly to 92%, the conidia activity decreased significantly, the size and complexity of conidia changed significantly, and the uptake and utilization rate of nitrogen source nutrients of the two knockout strains was lower than that of the wild strains. Ktr1 had no significant change in spore yield and colony growth, but the changes of Mannose Glycoprotein and chitin in cell wall were much greater than those of ktr4 and kre2. The mycelial cell wall became thinner and the hydrophobicity of spore cell wall decreased by 12%. ktr4 and kre2 cell wall alpha-glucan contents were significantly higher than those of wild type and ktrl spores. In general, ktr4 and kre2 were more sensitive to oxidative stress and cell wall interference than Aktrl, but their responses to hyperosmotic stress were not significantly different. The results showed that Ktr1, Ktr4 and Kre2 contributed to the biocontrol potential of Beauveria bassiana to varying degrees, but the contribution of Ktr4 and Kre2 was much greater than that of Ktr1. The acetylation of lysine in histone acetyltransferase Mst2 of Beauveria bassiana was closely related to the transcriptional activity of genes, but most of the acetyltransferases and deactivation of lysine were responsible for this. There is a histone acetyltransferase Mst2 in Beauveria bassiana, which is homologous to Schizosaccharomyces pombe acetyltransferase SpMst2 and can specifically acetylate group. In order to clarify the role of Mst2 in Beauveria bassiana, we constructed and analyzed a knockout/replenishment strain of Mst2. Phenotypic experiments showed that the ability of AMst2 to utilize different carbon and nitrogen sources was far inferior to that of wild and replenished strains. The ability of conidia of knockout plants to resist high temperature and ultraviolet radiation was significantly weakened. The surface hydrophobicity and infectivity to sensitive insects were all decreased. Cell flow analysis showed that AMst2 sporozoites became smaller, their density decreased, S phase prolonged and G2/M phase shortened. As a result, Mst2 plays a key role in DNA damage nodes and is involved in the regulation of cell cycle, asexual development, multiple stress responses and insect host infectivity of Beauveria bassiana. Different from the model filamentous fungi, Beauveria bassiana has only Fkh2, and there is no Fkhl. Knocking out the Fkh2 gene from wild strains results in the cell cycle disorder of Beauveria bassiana, which slows down the colony growth in different carbon and nitrogen sources, and knocks out the mycelial septum of the strains. Interestingly, the conidia yield of knockout strains was significantly higher than that of wild strains under normal plate culture conditions, but both spores were significantly smaller and less dense, indicating a decrease in spore contents. The increased susceptibility to osmotic stress and the decreased ability to tolerate UV-B radiation and high temperature suggest that Fkh2 may be involved in the stress response of the host bacteria. In addition, the bioassays of body wall infection and infection by injection of conidia suspension showed that the virulence of the knockout strain was significantly reduced. The results showed that Fkh2 not only participated in regulating cell cycle of Beauveria bassiana, but also regulated growth and development, multiple stress responses and virulence. Two 6-phosphorus compounds were found in Beauveria bassiana. Functional analysis of trehalose synthase homologues Trehalose biosynthesis pathways are important to plant and animal pathogenic fungi because intracellular trehalose accumulation levels are related to host bacteria'environmental adaptability and host infectivity. Beauveria bassiana has two 6-phosphate trehalose synthase (TPS), TpsA and TpsB, respectively. The construction and analysis of single gene, double gene knockout strain and complement strain showed that the activity of TPS and the accumulation of trehalose could not be detected in mycelial cells of double knockout strain tpsA tpsB, while the activity of TPS and trehalose accumulation of single knockout strain tpsA decreased by 71-75% and 72% respectively under normal and same stress conditions. TpsB decreased by 21-30% and 15-45% respectively. The sum of TPS enzyme activity loss or trehalose accumulation decrease of two single knocking strains under given conditions was close to that of double knocking strains. Interestingly, trehalose content, wall structure, hydrophobicity, vigor, size and density of spores were most severely impaired in the double knocking plants, followed by tpsA and tpsB. The same trend was observed in the three knocking out plants to oxidize, hyperosmotic, cell wall interference, high temperature and UV-B ultraviolet radiation. These results show that TpsA in Beauveria bassiana plays a more important role in regulating trehalose synthesis, vegetative growth, spore quality, multiple stress responses and host infection than TpsB, and the latter only shows a slightly stronger role in sporulation than the former. The regulatory effect of psA and TpsB on each phenotype of Beauveria bassiana is additive, which is different from the research reports on many TPS homologues in some model filamentous fungi. Therefore, two TPS homologous proteins have special effects on the adaptation of Beauveria bassiana to different types of insect hosts and their complex and diverse environments. Important significance.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S476.12

【共引文献】