發(fā)布時(shí)間：2018-09-15 05:28

【摘要】：球孢白僵菌(Beauveria bassiana)是廣泛用于農(nóng)林害蟲生物防治的昆蟲病原真菌,其侵染體是分生孢子,也是許多真菌殺蟲劑的活性成分。分生孢子制劑防治田間害蟲的效果往往受到溫度、濕度、紫外輻射及化學(xué)農(nóng)藥等多種環(huán)境脅迫的影響。因此,探索生防真菌抗逆境脅迫的生理機(jī)制,對(duì)以提高其生防潛能為目標(biāo)的菌株遺傳改良及菌劑的合理應(yīng)用具有重要意義。本研究圍繞這一目標(biāo),一是解析了球孢白僵菌甘露糖轉(zhuǎn)移酶PMT和Ktr家族的功能,揭示了它們對(duì)細(xì)胞生長發(fā)育、環(huán)境抗逆性及寄主侵染力的重要貢獻(xiàn)。二是研究解析了乙酰轉(zhuǎn)移酶Mst2對(duì)球孢白僵菌生長發(fā)育、脅迫應(yīng)答、細(xì)胞周期和毒力的影響。三是解析forkhead轉(zhuǎn)錄因子Fkh2的功能,發(fā)現(xiàn)Fkh2通過調(diào)節(jié)相關(guān)基因的轉(zhuǎn)錄而參與球孢白僵菌細(xì)胞周期、無性發(fā)育及毒力的調(diào)控。四是解析了球孢白僵菌中兩個(gè)海藻糖合成酶同源蛋白TpsA和TpsB對(duì)海藻糖合成、生長發(fā)育、孢子質(zhì)量、多脅迫響應(yīng)及毒力的貢獻(xiàn),發(fā)現(xiàn)二者對(duì)幾乎所有生防潛能相關(guān)性狀的貢獻(xiàn)都表現(xiàn)為加性效應(yīng)。主要的研究內(nèi)容和結(jié)果概述如下：球孢白僵菌PMT家族甘露糖轉(zhuǎn)移酶的功能解析PMT家族是在內(nèi)質(zhì)網(wǎng)上催化第一個(gè)甘露糖轉(zhuǎn)移到蛋白質(zhì)絲氨酸/蘇氨酸上的一類甘露糖轉(zhuǎn)移酶,根據(jù)結(jié)構(gòu)特征可分為PMT1、PMT2和PMT4三個(gè)亞族。這三個(gè)亞族都存在于球孢白僵菌中且每個(gè)亞族只有一個(gè)成員,分別為Pmtl、Pmt2和Pmt4。其中Pmt2是敲除致死的,通過構(gòu)建Pmt2的RNA干擾菌株和Pmtl及Pmt4的單基因敲除/回補(bǔ)菌株并進(jìn)行多種表型分析,三個(gè)基因的功能得到較為詳盡的解析。與野生型相比,單基因缺失或RNA干擾菌株在生長、產(chǎn)孢、孢子活力、多脅迫耐受力及毒力等諸方面均表現(xiàn)不同程度的缺陷。其中,Pmt2的三個(gè)干擾菌株、APmtl及ΔPmt4在富營養(yǎng)和限制培養(yǎng)基上的生長減慢20～79%,分生孢子產(chǎn)量下降16～72%,并伴隨孢子活力顯著下降即萌發(fā)一半所需的時(shí)間顯著延長。不僅如此,干擾及敲除菌株在菌絲生長或孢子萌發(fā)期間,對(duì)氧化、高滲、胞壁干擾、高溫及UV-B輻射等環(huán)境脅迫的抵抗力都有不同程度的顯著下降。在對(duì)大蠟螟(Galleria mellonella)幼蟲的生物測(cè)定中,突變株經(jīng)正常體壁侵染的毒力下降53～62%,但通過血腔注射的毒力未發(fā)生大的變化。值得一提的是,突變株的孢壁完整性遭受很大程度的破壞,包括孢壁變薄、孢子表面疏水性降低及重要孢壁成分發(fā)生改變。所有表型的變化都在回補(bǔ)株中得到恢復(fù)。因此,PMT家族在球孢白僵菌中都不是功能冗余的,而是各成員相互協(xié)調(diào)共同調(diào)節(jié)宿主菌對(duì)復(fù)雜多樣環(huán)境的適應(yīng)性及對(duì)昆蟲寄主的侵染力,因而在球孢白僵菌生防潛能的維持中發(fā)揮著互不替代的重要作用。球孢白僵菌Ktr家族甘露糖轉(zhuǎn)移酶的功能比較Ktr家族甘露糖轉(zhuǎn)移酶的功能是將第二個(gè)甘露糖連接到被PMT家族成員轉(zhuǎn)移的第一個(gè)甘露糖基上,因而是對(duì)糖蛋白甘露糖鏈進(jìn)行延伸的轉(zhuǎn)移酶,其調(diào)控的反應(yīng)發(fā)生在高爾基體內(nèi)。球孢白僵菌有三個(gè)Ktr家族成員,分別為Ktrl、Ktr4和Kre2/Mnt1。通過單基因敲除/回補(bǔ)菌株的構(gòu)建并進(jìn)行表型分析,發(fā)現(xiàn)Δktr4和Δkre2的分生孢子產(chǎn)量大幅下降～92%,孢子活力顯著降低,孢子大小和復(fù)雜度也發(fā)生顯著變化；兩個(gè)敲除菌株對(duì)培養(yǎng)基中不同碳、氮源營養(yǎng)的攝入利用率不如野生株,表現(xiàn)為菌落生長緩慢。與野生型相比,Δktr1在孢子產(chǎn)量及菌落生長方面并無顯著變化,但細(xì)胞壁中甘露糖蛋白和幾丁質(zhì)等成分的變化都遠(yuǎn)大于Δktr4和Δkre2,菌絲細(xì)胞壁變薄,孢子細(xì)胞壁表面疏水性下降12%。Δktr4和Δkre2細(xì)胞壁中α-葡聚糖含量顯著高于野生型和Δktrl,孢子壁厚度也比Δktr1更薄,孢子表面疏水性分別下降64%和71%�？傮w上,Δktr4和Δkre2比Aktrl對(duì)氧化和胞壁干擾脅迫更敏感,但三者對(duì)高滲脅迫的反應(yīng)則差異不大。高溫、UV-B輻射耐受性以及毒力在Δktr4和Δkre2中都大大下降,而Δktrl只對(duì)高溫敏感。結(jié)果顯示,Ktr1、Ktr4和Kre2對(duì)球孢白僵菌生防潛能都有不同程度的貢獻(xiàn),但Ktr4和Kre2的貢獻(xiàn)遠(yuǎn)大于Ktr1的貢獻(xiàn)。球孢白僵菌組蛋白乙酰轉(zhuǎn)移酶Mst2的功能解析組蛋白中賴氨酸的乙�；c基因的轉(zhuǎn)錄活性密切相關(guān),但由于多數(shù)乙酰轉(zhuǎn)移酶和去乙酰轉(zhuǎn)移酶對(duì)賴氨酸的特異性修飾作用知之有限,每個(gè)賴氨酸乙�；c細(xì)胞功能之間的關(guān)系目前并不十分清楚。球孢白僵菌中有一個(gè)組蛋白乙酰轉(zhuǎn)移酶Mst2,它與裂殖酵母(Schizosaccharomyces pombe)乙酰轉(zhuǎn)移酶SpMst2同源,并能特異性乙�；M蛋白3(H3)的第14位賴氨酸(H3K14)。為了明確Mst2在球孢白僵菌中的作用,我們構(gòu)建和分析了Mst2的敲除／回補(bǔ)菌株。表型實(shí)驗(yàn)顯示,AMst2對(duì)不同碳氮源的利用能力遠(yuǎn)不如野生株和回補(bǔ)株,正常培養(yǎng)條件下的產(chǎn)孢能力受損較大,孢子活力下降,抵抗氧化、高滲及胞壁干擾脅迫的能力顯著下降。敲除株分生孢子耐高溫、抗紫外能力顯著減弱,表面疏水性和對(duì)敏感昆蟲的侵染力均下降。細(xì)胞流式分析顯示,AMst2的芽生孢子變小,密度降低,細(xì)胞周期中S期延長,G2/M期縮短。綜合所有分析結(jié)果,Mst2在DNA損傷節(jié)點(diǎn)上起著關(guān)鍵作用,因而參與調(diào)控球孢白僵菌的細(xì)胞周期、無性發(fā)育、多脅迫應(yīng)答及其對(duì)昆蟲寄主的侵染力。球孢白僵菌叉頭轉(zhuǎn)錄因子Fkh2的功能解析模式絲狀真菌一般擁有叉頭Forkhead (FKH)轉(zhuǎn)錄因子Fkhl和Fkh2,主要參與細(xì)胞周期的調(diào)控而影響生物學(xué)性狀。不同于模式絲狀真菌,球孢白僵菌只有Fkh2,不存在Fkhl。將Fkh2編碼基因從野生株中敲除,導(dǎo)致球孢白僵菌細(xì)胞周期發(fā)生紊亂,在不同碳氮源培養(yǎng)基上的菌落生長不同程度地減慢,而且敲除菌株的菌絲隔膜增多,菌絲細(xì)胞粗短。有趣的是,敲除株的產(chǎn)孢提前,在正常平板培養(yǎng)條件下分生孢子產(chǎn)量顯著升高,在液培條件下芽生孢子的產(chǎn)量也顯著高于野生株,但兩種孢子都明顯變小,且密度降低,顯示孢子內(nèi)含物減少。其分生孢子對(duì)氧化和高滲脅迫的敏感性升高,耐UV-B輻射和高溫的能力減弱,說明Fkh2可能參與宿主菌的脅迫應(yīng)答。此外,用分生孢子懸液對(duì)大蠟螟幼蟲進(jìn)行體壁侵染和注射侵染的生物測(cè)定,結(jié)果顯示敲除菌株的毒力顯著降低。敲除菌株所有這些表型變化都在若干表型相關(guān)功能基因的轉(zhuǎn)錄分析中獲得支持,并且都在回補(bǔ)菌株中得到很好的恢復(fù)。結(jié)果顯示,Fkh2不僅參與調(diào)控球孢白僵菌的細(xì)胞周期循環(huán),而且還調(diào)控生長發(fā)育、多脅迫應(yīng)答及毒力等多種生防潛能相關(guān)的性狀。球孢白僵菌中兩種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徑對(duì)于動(dòng)植物病原真菌是非常重要的,因?yàn)榧?xì)胞內(nèi)海藻糖的積累水平關(guān)系到宿主菌的環(huán)境適應(yīng)性和寄主侵染力。球孢白僵菌有兩個(gè)6-磷酸海藻糖合成酶(TPS),分別為TpsA和TpsB。通過單基因、雙基因敲除菌株及回補(bǔ)菌株的構(gòu)建與分析,發(fā)現(xiàn)雙敲菌株△tpsA△tpsB的菌絲細(xì)胞中既檢測(cè)不到TPS的酶活,也檢測(cè)不到任何海藻糖的積累；而單敲菌株△tpsA的TPS酶活和海藻糖積累水平在正常和同脅迫條件下分別下降71～75%和72～80%,在△tpsB中分別下降21～30%和15～45%。兩個(gè)單敲株在給定條件下TPS酶活損失或海藻糖積累水平下降幅度之和,正好接近雙敲菌株的酶活損失或海藻糖含量的下降幅度。正常培養(yǎng)條件下分生孢子產(chǎn)量在雙敲株中下降達(dá)98%,而在△tpsA和△tpsB中分別下移33%和50%。有趣的是,表征孢子質(zhì)量的海藻糖含量、孢壁結(jié)構(gòu)成分、疏水性、活力、大小及密度均在雙敲株中受損最嚴(yán)重,后依次為△tpsA和△tpsB。相同的趨勢(shì)也見于三個(gè)敲除株對(duì)氧化、高滲、胞壁干擾、高溫及UV-B紫外輻射等環(huán)境脅迫抵抗力的缺陷變化以及對(duì)大蠟螟幼蟲毒力的缺陷變化。這些結(jié)果證明,球孢白僵菌中TpsA對(duì)海藻糖合成、營養(yǎng)生長、孢子質(zhì)量、多脅迫應(yīng)答及寄主侵染過程的調(diào)控作用均大于TpsB,后者僅表現(xiàn)比前者稍強(qiáng)的產(chǎn)孢調(diào)控作用。最重要的是,TpsA和TpsB對(duì)球孢白僵菌每種表型的調(diào)控作用都表現(xiàn)為加性效應(yīng),不同于一些模式絲狀真菌中多個(gè)TPS同源物不一定都起作用的研究報(bào)道。因此,兩個(gè)TPS同源蛋白對(duì)球孢白僵菌適應(yīng)不同類型昆蟲寄主及其復(fù)雜多樣環(huán)境的生存方式具有特別重要的意義。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S476.12

【共引文獻(xiàn)】

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