渗透胁迫下sakA/hogA基因对黄曲霉毒素合成及致病性的影响

论文摘要

黄曲霉是腐生真菌,属于曲霉属。这种真菌是一种机会致病菌,能够污染大多数种子作物和其他生物,包括动物和人类。更严重的是,黄曲霉由于其次级代谢产物的高毒性而导致死亡或慢性疾病。因此,影响黄曲霉生长和分布的各种因素、黄曲霉毒素生物合成途径、基因簇以及调控机制已成为过去几十年研究的主题,其中包括响应于渗透压的（MAPK/HOG）途径促分裂原活化蛋白激酶信号的研究。尽管该途径已在一些真菌中广泛研究,但在黄曲霉中,其机制尚不清楚。Sak A（hog1/hogA）被认为是MAPK/HOG途径的重要部分,为此本研究重点是确定Sak A在黄曲霉中的生物学功能。在本研究中,通过同源重组的方法进行敲除和互补菌株的构建,随后通过1.2mo L/L的氯化钠（NaCl）、1.2 mo L/L D-山梨醇、以及0.93和0.99水活度诱导渗透压。实验发现,与野生型（WT）和互补菌株（ΔAfsakA::AfsakA）相比,基因敲除株（ΔAfsakA）对分生孢子产生显著影响。q RT-PCR结果显示,aba A和brl A基因的表达水平在ΔAfsakA中,比WT和ΔAfsakA::AfsakA菌株中发生下调。研究还发现,Af Sak A对细胞壁应激压力和渗透压都有响应。渗透压相关基因HPS,GPD,GRE和STL的转录水平在ΔAfsakA中,显著低于WT和ΔAfsakA::AfsakA菌株。在没有渗透胁迫的情况下,ΔAfsakA突变体比他菌株产生更多的菌核;而在渗透胁迫存在下,所有菌株都不能产生任何菌核。与菌核生产相关基因（nsdC和nsdD）的转录水平研究表明,在没有胁迫的情况下,这两个基因在ΔAfsakA中的表达水平高于WT和ΔAfsakA::AfsakA菌株。对于黄曲霉毒素的研究表明,ΔAfsakA突变体在0.93aw渗透胁迫条件下会抑制黄曲霉毒素B1（AFB1）的产生。然而,在1.2mo L/L D-山梨醇、0.93aw和0.99aw渗透胁迫条件下,Af Sak A的缺失导致AFB1产量的显着增加,这与WT和ΔAfsakA::AfsakA菌株不同。在没有应激的情况下,通过qRTPCR进一步检测黄曲霉毒素合成基因（aflO和aflQ）和黄曲霉毒素转录调节基因（aflR和aflS）的表达水平。结果显示,与WT和ΔAfsakA::AfsakA菌株相比,ΔAfsakA菌株中所有这些黄曲霉毒素合成基因和调节基因的表达量均上调。对花生种子和玉米籽粒进行致病性试验表明,ΔAfsakA菌株比WT和ΔAfsakA::AfsakA菌株产生更多的AFB1和分生孢子。亚细胞定位结果证明,在没有渗透压刺激的情况下,AfSakA-m Cherry位于细胞质中,而在渗透压力刺激时它会移位至细胞核。总之,本研究提供了关于黄曲霉毒素生物合成与调控的新见解,同时为防治黄曲霉对农产品、生物和人类造成的持续感染以及调控黄曲霉对环境的污染,提供了一定的理论基础。

论文目录

• Appendix list of abbreviations
• 摘要
• Abstract
• 1 Introduction
•   1.1 Generalities of Aspergillus flavus
•   1.2 Sexual and asexual development of Aspergillus flavus
•   1.3 Diseases and epidemiological cycles of Aspergillus flavus
•   1.4 Spreading methods and preventive techniques of Aspergillus flavus
•     1.4.1 Spreading methods of Aspergillus flavus
•     1.4.2 Preventive techniques of Aspergillus flavus
•   1.5 Overview of major aflatoxins
•   1.6 Genomic and aflatoxin biosynthetic in Aspergillus flavus
•   1.7 The mitogen activated protein kinases and their cascades pathways
•   1.8 High osmolarity/glycerol （HOG-MAPK） pathway
•   1.9 The mitogen activated protein kinase SakA/Hog1
•   1.10 Purpose and significance of the study
• 2 Material and methods used in the study
•   2.1 Research materials
•     2.1.1 Strains used in the study
•     2.1.2 Primers used in the study
•     2.1.3 Instruments and reagents used in the study
•     2.1.4 Instruments and reagents used in the study
•   2.2 Research methodology
•     2.2.1 Bioinformatics analysis of MAPK SakA in Aspergillus flavus
•     2.2.2 Construction of the gene deletion and complementation mutant strains
•     2.2.3 Preparation of protoplast
•     2.2.4 The extraction of DNA in Aspergillus flavus
•     2.2.5 Polymeric chain reaction （PCR）
•     2.2.6 The transformation of protoplasts
•     2.2.7 Verification of the transformants and preparation of the spores
•     2.2.8 Preparation of agarose gel
•     2.2.9 The purification of DNA
•     2.2.10 Culture conditions for growth and analysis of sensitivity to stress
•     2.2.11 Analysis of conidia production
•     2.2.12 Analysis of conidiophores production
•     2.2.13 Analysis of sclerotia production
•     2.2.14 The production of aflatoxin B1 by Aspergillus flavus
•     2.2.15 The pathogenicity test
•     2.2.16 The extraction of RNA in Aspergillus flavus
•     2.2.17 The synthesis of c DNA from the total RNA
•     2.2.18 The qRT-PCR analysis
•     2.2.19 SakA subcellular localization in Aspergillus flavus
•     2.2.20 Statistical analysis
• 3 Results and analysis
•   3.1 Bioinformatics analysis of sakA gene in Aspergillus flavus
•   3.2 Construction of Aspergillus flavus sakA deletion （?AfsakA） and complementary（?AfsakA::AfsakA） strains
•   3.3 Effects of ?AfsakA on growth and its response to osmotic stress in Aspergillus flavus
•   3.4 Effect of ΔAfsakA on germination of conidia in Aspergillus flavus
•   3.5 Effect of AfsakA deletion on sclerotia production in Aspergillus flavus
•   3.6 Effect of ΔAfsakA on aflatoxin B1 （AFB1） production in Aspergillus flavus
•   3.7 Effects of ΔAfsakA on sensitivity to multiple stress in Aspergillus flavus
•   3.8 Effect of ΔAfsakA on virulence to crop seeds in Aspergillus flavus
•   3.9 Analysis of subcellular localization of AfSakA in Aspergillus flavus
• 4 Discussion
•   4.1 SakA gene in Aspergillus flavus
•   4.2 The role of AfsakA in asexual development with/without osmotic stress
•   4.3 The role of AfsakA in sexual development with/without osmotic stress
•   4.4 The role of AfsakA in osmotic stress response
•   4.5 The role of AfsakA in multiple stress response
•   4.6 The role of AfsakA in aflatoxin B1 biosynthesis
•   4.7 The role of AfsakA on pathogenicity
•   4.8 The subcellular localization of Af SakA in Aspergillus flavus
•   4.9 The role of sakA and its regulatory mechanism in pathogenicity of Aspergillus flavus
• 5. Conclusion
• References
• Published papers and other Academic achievements during Master degree program
• Short version of the thesis in Chinese
• Acknowledgement

文章来源

类型: 硕士论文

作者: Elisabeth Tumukunde

导师: 汪世华,袁军

关键词: 黄曲霉,渗透胁迫,促分裂原活化蛋白激酶途径,黄曲霉毒素

来源: 福建农林大学

年度: 2019

分类: 基础科学

专业: 生物学,生物学

单位: 福建农林大学

分类号: Q933

总页数: 123

文件大小: 3755K

下载量: 36