球孢白僵菌适应环境pH变化及胞内pH稳态维持的遗传分子基础及其对生物防治潜能的贡献

发布时间：2018-05-26 00:12

本文选题：昆虫病原真菌 + 球孢白僵菌　；参考：《浙江大学》2016年博士论文

【摘要】：球孢白僵菌(Beauveria bassiana)是广泛用于农林害虫生物防治的一种丝状昆虫病原真菌,其生物防治潜能不仅取决于毒力,而且取决于抗逆境能力。活体真菌制剂在田间应用时,不可避免地会暴露在各种类型的环境胁迫之下,如田间高温、紫外辐射、化学农药及土壤酸碱度等,这些都是影响菌剂杀虫效果和稳定性的重要因素。真菌许多重要的生命活动受环境pH的调控,而且细胞内pH稳态的维持对病原真菌的生长发育及致病性至关重要。本论文围绕球孢白僵菌如何适应环境pH变化这一主题,研究和解析了Pal信号通路的7个蛋白、Na+/H+逆向转运蛋白Nhx1及液泡ATP激酶H亚基VmaH的生物学功能,重点揭示它们编码的基因对球孢白僵生物防治潜能的贡献。主要研究内容和结果简述如下：Pal信号通路依赖pH调控球孢白僵菌的生长发育及高渗应答丝状真菌适应环境pH的变化主要受控于Pal信号通路。在球孢白僵菌中,Pal通路由转录因子PacC及上游六个Pal蛋白(PalA/B/C/F/H/I)组成。编码这些蛋白的所有基因在pH 9的碱性条件下转录表达水平最高。pacC及各pal的单基因敲除株在初始pH 7.3的环境的培养条件下,培养基的酸化显著延迟,并伴随着胞内外有机酸含量的改变。在碱性条件下的NaC1、KC1、LiCl或山梨醇的高渗胁迫条件下,除Δpall之外所有敲除株的生长受到显著抑制,而且碱性环境的高渗胁迫使其菌丝细胞内的液泡发生碎裂和形态的改变,即形成不规则的碎片状液泡。所有敲除株在pH 3条件下的分生孢子产量高于野生株,大多数敲除株在pH 7条件下的产孢量高于野生株,但在pH 9下的产孢量却低于野生株,并且在pH 9条件下所产分生孢子的大小和密度发生改变。所有敲除株对大蜡螟幼虫的毒力都有所降低。结果表明,Pal信号通路以依赖pH的方式调控球孢白僵菌的营养生长,产孢及高渗敏感性,而且对其毒力有显著贡献。球孢白僵菌Na+/H+逆向转运蛋白Nhx1的功能解析球孢白僵菌胞内只有一个NHE家族成员,即Na+/H+逆向转运蛋白Nhx1,对其的研究在丝状真菌中罕见报道。将nhx1与eGFP融合后在野生菌株中表达,并利用荧光显微镜观察可见融合蛋白分布在菌丝细胞隔膜和管状液泡之间的点状结构上,但确定不定位于液泡中。根据酿酒酵母Nhx1与其特殊细胞器上的标记蛋白共定位的研究报道推测,上述点状结构可能是类似酵母细胞内的小体和跨高尔基体网络。nhx1的缺失导致胞内液泡酸化和液泡整合受阻或裂解加剧,表现为菌丝细胞内形成很多小而不规则的液泡；而野生菌株胞内液泡呈球状或管状,既大且少。敲除株的气生分生孢子和液生芽生孢子的产量剧幅下降。其分生孢子在不同碳氮源的基础培养基和富营养培养基上的萌发速率较为缓慢,并且其菌落生长也表现出不同程度的缺陷。敲除株对高渗、高温及部分金属离子胁迫的敏感性升高,但分生孢子抗UV-B辐射的能力却显著增强。令人感兴趣的是,敲除株不能经昆虫体壁或血腔注射侵染大蜡螟幼虫,即其致病力完全丧失。进一步研究发现,敲除株在培养期间分泌至胞外的体壁降解酶的活力大幅下降；注射到昆虫血腔的分生孢子不能形成芽生孢子。敲除株所有的这些变化在回补株中均得到恢复。研究结果表明,Nhx1不仅参与调节液泡内pH平衡和液泡的形态,而且也是球孢白僵菌在昆虫体外或体内的无性循环所必不可少的。球孢白僵菌液泡ATP激酶亚基H的功能解析VmaH是真菌液泡ATP激酶复合体的众多亚基之一。将球孢白僵菌的vmaH同源基因与绿色荧光蛋白基因eGFP融合并转入野生株中表达后,证明VmaH定位于细胞质而不是液泡中。vmaH的缺失导致菌丝液泡内pH显著上升,而培养液的酸化加快,并伴随分泌到胞外的氨含量急剧下降87%。由于细胞内外pH的失衡,敲除株在不同碳氮源的基础培养基和富营养培养基上,从酸性至中性条件下的生长有不同程度缺陷,但在碱性条件下生长缺陷缩小敲除株在正常培养条件下的产孢大幅延迟,但最终产孢量仅下降10%左右；液体培养的芽生孢子产量显著下降,并且所产气生分生孢子和液生芽生孢子的形态大小和密度也发生显著变化。敲除株对大蜡螟幼虫的毒力显著下降,而且经体壁侵染的毒力降幅大于经血腔注射侵染的毒力降幅,其分生孢子耐高温、抗紫外辐射的能力也显著下降。结果表明,VmaH是维持球孢白僵菌细胞内外pH稳态的重要因子,因而对其生长发育、产孢、毒力及抗逆力等生防潜能相关性状具有显著的贡献。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476.12

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