本文選題：昆蟲病原真菌 + 球孢白僵菌��； 參考：《浙江大學(xué)》2016年博士論文

【摘要】：球孢白僵菌(Beauveria bassiana)是廣泛用于農(nóng)林害蟲生物防治的一種絲狀昆蟲病原真菌,其生物防治潛能不僅取決于毒力,而且取決于抗逆境能力�；铙w真菌制劑在田間應(yīng)用時,不可避免地會暴露在各種類型的環(huán)境脅迫之下,如田間高溫、紫外輻射、化學(xué)農(nóng)藥及土壤酸堿度等,這些都是影響菌劑殺蟲效果和穩(wěn)定性的重要因素。真菌許多重要的生命活動受環(huán)境pH的調(diào)控,而且細(xì)胞內(nèi)pH穩(wěn)態(tài)的維持對病原真菌的生長發(fā)育及致病性至關(guān)重要。本論文圍繞球孢白僵菌如何適應(yīng)環(huán)境pH變化這一主題,研究和解析了Pal信號通路的7個蛋白、Na+/H+逆向轉(zhuǎn)運蛋白Nhx1及液泡ATP激酶H亞基VmaH的生物學(xué)功能,重點揭示它們編碼的基因?qū)η蜴甙捉┥锓乐螡撃艿呢暙I(xiàn)。主要研究內(nèi)容和結(jié)果簡述如下：Pal信號通路依賴pH調(diào)控球孢白僵菌的生長發(fā)育及高滲應(yīng)答絲狀真菌適應(yīng)環(huán)境pH的變化主要受控于Pal信號通路。在球孢白僵菌中,Pal通路由轉(zhuǎn)錄因子PacC及上游六個Pal蛋白(PalA/B/C/F/H/I)組成。編碼這些蛋白的所有基因在pH 9的堿性條件下轉(zhuǎn)錄表達(dá)水平最高。pacC及各pal的單基因敲除株在初始pH 7.3的環(huán)境的培養(yǎng)條件下,培養(yǎng)基的酸化顯著延遲,并伴隨著胞內(nèi)外有機(jī)酸含量的改變。在堿性條件下的NaC1、KC1、LiCl或山梨醇的高滲脅迫條件下,除Δpall之外所有敲除株的生長受到顯著抑制,而且堿性環(huán)境的高滲脅迫使其菌絲細(xì)胞內(nèi)的液泡發(fā)生碎裂和形態(tài)的改變,即形成不規(guī)則的碎片狀液泡。所有敲除株在pH 3條件下的分生孢子產(chǎn)量高于野生株,大多數(shù)敲除株在pH 7條件下的產(chǎn)孢量高于野生株,但在pH 9下的產(chǎn)孢量卻低于野生株,并且在pH 9條件下所產(chǎn)分生孢子的大小和密度發(fā)生改變。所有敲除株對大蠟螟幼蟲的毒力都有所降低。結(jié)果表明,Pal信號通路以依賴pH的方式調(diào)控球孢白僵菌的營養(yǎng)生長,產(chǎn)孢及高滲敏感性,而且對其毒力有顯著貢獻(xiàn)。球孢白僵菌Na+/H+逆向轉(zhuǎn)運蛋白Nhx1的功能解析球孢白僵菌胞內(nèi)只有一個NHE家族成員,即Na+/H+逆向轉(zhuǎn)運蛋白Nhx1,對其的研究在絲狀真菌中罕見報道。將nhx1與eGFP融合后在野生菌株中表達(dá),并利用熒光顯微鏡觀察可見融合蛋白分布在菌絲細(xì)胞隔膜和管狀液泡之間的點狀結(jié)構(gòu)上,但確定不定位于液泡中。根據(jù)釀酒酵母Nhx1與其特殊細(xì)胞器上的標(biāo)記蛋白共定位的研究報道推測,上述點狀結(jié)構(gòu)可能是類似酵母細(xì)胞內(nèi)的小體和跨高爾基體網(wǎng)絡(luò)。nhx1的缺失導(dǎo)致胞內(nèi)液泡酸化和液泡整合受阻或裂解加劇,表現(xiàn)為菌絲細(xì)胞內(nèi)形成很多小而不規(guī)則的液泡；而野生菌株胞內(nèi)液泡呈球狀或管狀,既大且少。敲除株的氣生分生孢子和液生芽生孢子的產(chǎn)量劇幅下降。其分生孢子在不同碳氮源的基礎(chǔ)培養(yǎng)基和富營養(yǎng)培養(yǎng)基上的萌發(fā)速率較為緩慢,并且其菌落生長也表現(xiàn)出不同程度的缺陷。敲除株對高滲、高溫及部分金屬離子脅迫的敏感性升高,但分生孢子抗UV-B輻射的能力卻顯著增強(qiáng)。令人感興趣的是,敲除株不能經(jīng)昆蟲體壁或血腔注射侵染大蠟螟幼蟲,即其致病力完全喪失。進(jìn)一步研究發(fā)現(xiàn),敲除株在培養(yǎng)期間分泌至胞外的體壁降解酶的活力大幅下降；注射到昆蟲血腔的分生孢子不能形成芽生孢子。敲除株所有的這些變化在回補株中均得到恢復(fù)。研究結(jié)果表明,Nhx1不僅參與調(diào)節(jié)液泡內(nèi)pH平衡和液泡的形態(tài),而且也是球孢白僵菌在昆蟲體外或體內(nèi)的無性循環(huán)所必不可少的。球孢白僵菌液泡ATP激酶亞基H的功能解析VmaH是真菌液泡ATP激酶復(fù)合體的眾多亞基之一。將球孢白僵菌的vmaH同源基因與綠色熒光蛋白基因eGFP融合并轉(zhuǎn)入野生株中表達(dá)后,證明VmaH定位于細(xì)胞質(zhì)而不是液泡中。vmaH的缺失導(dǎo)致菌絲液泡內(nèi)pH顯著上升,而培養(yǎng)液的酸化加快,并伴隨分泌到胞外的氨含量急劇下降87%。由于細(xì)胞內(nèi)外pH的失衡,敲除株在不同碳氮源的基礎(chǔ)培養(yǎng)基和富營養(yǎng)培養(yǎng)基上,從酸性至中性條件下的生長有不同程度缺陷,但在堿性條件下生長缺陷縮小敲除株在正常培養(yǎng)條件下的產(chǎn)孢大幅延遲,但最終產(chǎn)孢量僅下降10%左右；液體培養(yǎng)的芽生孢子產(chǎn)量顯著下降,并且所產(chǎn)氣生分生孢子和液生芽生孢子的形態(tài)大小和密度也發(fā)生顯著變化。敲除株對大蠟螟幼蟲的毒力顯著下降,而且經(jīng)體壁侵染的毒力降幅大于經(jīng)血腔注射侵染的毒力降幅,其分生孢子耐高溫、抗紫外輻射的能力也顯著下降。結(jié)果表明,VmaH是維持球孢白僵菌細(xì)胞內(nèi)外pH穩(wěn)態(tài)的重要因子,因而對其生長發(fā)育、產(chǎn)孢、毒力及抗逆力等生防潛能相關(guān)性狀具有顯著的貢獻(xiàn)。
[Abstract]:Beauveria bassiana is a filamentous insect pathogenic fungus widely used in the biological control of agroforestry pests. Its biological control potential depends not only on virulence but also on the ability to resist adversity. In the field, living fungal agents are inevitably exposed to various types of environmental stresses, such as high temperature in the field, and purple. External radiation, chemical pesticides and soil acidity and alkalinity are all important factors affecting the insecticidal effect and stability of the fungi. Many important biological activities of fungi are regulated by the environment pH, and the maintenance of pH homeostasis in the cells is very important to the growth and pathogenicity of the pathogenic fungi. This paper focuses on how the Bacillus bassiana adapts to the environment pH In this subject, the biological functions of 7 proteins of Pal signaling pathway, Na+/H+ reverse transporter Nhx1 and vacuolar ATP kinase H subunit VmaH are studied and analyzed. The contribution of their encoded genes to the biocontrol potential of ospora ospora is highlighted. The main contents and results are as follows: the Pal signaling pathway relies on pH to regulate the sporulation white. The growth and development of mycelia and hypertonic response to the adaptation of filamentous fungi to the environment pH are mainly controlled by the Pal signaling pathway. In the Bacillus bassiana, the Pal route transcription factor PacC and the upstream six Pal protein (PalA/B/C/F/H/I) are composed. All genes encoding these proteins are at the highest level.PacC and each pal at the alkaline condition of pH 9. Under the conditions of the initial pH 7.3 environment, the acidification of the medium is significantly delayed and the content of the organic acids and acids inside and outside the cell are changed. Under the hyperosmotic stress of NaC1, KC1, LiCl or sorbitol under alkaline conditions, the growth of all the knockout strains except delta pall is significantly inhibited and the alkaline environment is hypertonic. The vacuoles of the vacuoles in the mycelial cells were forced to form a fragmentation and morphological change, namely the formation of an irregular fragment like vacuole. The yield of the conidia of all the knockout strains under the condition of pH 3 was higher than that of the wild plant. Most of the knockout strains were higher than the wild plants under the condition of pH 7, but the spore yield under pH 9 was lower than that of the wild plant, and under the condition of pH 9. The size and density of the conidia were changed. The toxicity of all the knockout strains to the larva of the paraffin borer was reduced. The results showed that the Pal signaling pathway regulated the vegetative growth, sporulation and hypertonic sensitivity of Beauveria bassiana by means of pH, and had significant contribution to its virulence, and the Na+/H+ reverse transporter Nhx1 of Beauveria bassiana. Only one member of the NHE family of Beauveria bassiana, Na+/H+ reverse transporter Nhx1, is rarely reported in filamentous fungi. NHX1 and eGFP are fused in the wild strains, and the spot structure between the membrane and tubular vacuoles of the filamentous membrane and the tubular vacuoles is observed by the fluorescence microscope. According to the study of the co localization of the Saccharomyces cerevisiae Nhx1 and the marker protein on its special organelles, it is suggested that the deletion of the dot like structure may be the loss of intracellular vacuole acidification and vacuolation of the intracellular vacuole and the vacuolar integration, which may be caused by the absence of.Nhx1 in the yeast cells. Many small and irregular vacuoles were formed in the cells, while the vacuoles in the wild strains were bulbous or tubular, both large and small. The output of the spore and the spores of the plant were decreased. The germination rate of the conidium on the basal medium and the eutrophic culture medium of different carbon and nitrogen sources was slower, and the colony of the conidia was the colony. Growth also showed a different degree of defect. The sensitivity of the knockout strains to hypertonic, high temperature and partial metal ion stress increased, but the ability of conidia to resist UV-B radiation was significantly enhanced. It is interesting that the knockout plant can not infect young insects of the large wax borer by injecting the insect body wall or blood cavity. The activity of the parietal degrading enzymes secreted to the extracellular during the incubation period was greatly reduced; the conidia injected into the insect's blood cavity did not form the spores. All these changes were restored in the remedial plants. The results showed that Nhx1 was not only involved in the regulation of pH balance and the form of vacuoles in the liquid bubble, but also the ball. The function of ATP kinase subunit H of Beauveria bassiana is one of the subunits of the fungal vacuoles ATP kinase complex, which is essential for the asexual cycle of the vesicles in vitro or in the body. The vmaH homologous gene of Beauveria bassiana and the green fluorescent protein gene eGFP are fused and transferred into the wild strain to prove that VmaH is determined by VmaH. The deletion of.VmaH in the cytoplasm rather than the vacuoles leads to a significant increase in the pH in the mycelial vacuoles, while the acidification of the culture fluid is accelerated and the ammonia content in the extracellular secretes a sharp decrease in 87%. due to the imbalance of pH in the cell and in the rich culture medium with different carbon and nitrogen sources, from acidic to neutral conditions. There were different degrees of defects, but the sporulation of the knockout plant under normal conditions was delayed significantly under normal conditions, but the final sporulation decreased by only about 10%, and the yield of the sprout spores in the liquid culture decreased significantly, and the morphology and density of the spore and the liquid spore were also significantly changed. The virulence of the knockout strain on the larva of the paraffin borer was significantly decreased, and the virulence of the infection by the body wall was greater than that in the blood cavity, and the conidium was resistant to high temperature and the ability to resist ultraviolet radiation significantly decreased. The results showed that VmaH was an important factor in maintaining the homeostasis of pH in the cells inside and outside of Beauveria bassiana. Spores, virulence and resilience have significant contributions to the correlation of biocontrol potential.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S476.12

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