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目录>
  • 封面
    答辩决议书
    中文摘要
    英文摘要
    目录
    Abbreviations
    Chapter 1: Literature Review
    1. Molecular mechanism of plants autophagy
    2. Autophagy and Senescence
    3. The innate immune system in plants
    4. Role of autophagy in plants immunity
    5. An overview of selective autophagy
    6. Research progress of virus-induced gene silencing system
    7. The significance and major content of this study
    Chapter 2: Materials and methods
    1 Experimental materials
    1.1 Plant material
    1.2 Strain materials
    1.3 Experimental reagents and instruments
    1.4 Experimental design softwares
    2. Experimental methods
    2.1 Cultivation of soybean plants
    2.2 Construction of BPMV-GmATG2, BPMV-GmATG5, and BPMV-GmATG7, silencing vectors
    2.3 Obtaining BPMV-GmATG2, BPMV-GmATG5 and BPMV-GmATG7 si-lenced strains by particles bombardment
    2.4 Silenced Plants Phenotype and molecular verification
    2.5 DAB staining
    2.6 Extraction of salicylic acid
    2.7 Analysis of disease resistance of silenced plants
    2.8 SMV-GUS infects soybean plants
    2.9 Analysis of kinase activation by western blotting assay
    2.10 Western blot analysis of autophagy-related proteins accumulation
    Chapter 3: Experimental Results andAnalysis
    1. Silencing autophagy-related genes in soybean results in accelerated senescence phenotypes under dark conditions
    1.1 Molecular verification of silencing effects by RT-PCR
    1.2 Accelerated senescence was observed in the GmATG2-, GmATG5- and GmATG7- silenced plants under dark treatments
    2. Silencing GmATG2, GmATG5 and GmATG7,respectively, results in activated defense responses
    2.1 Increased accumulation of H2O2
    2.2 Increased accumulation of the bound SA and highly induced expres-sion of PR1 in response to dark treatment in the GmATG2-, GmATG5- and GmATG7-silenced plants
    3. Silencing GmATG2, GmATG5 and GmATG7 results in enhanced re-sistance to Pseudomonas syringae pv. glycinea (Psg) and Soybean mo-saic virus (SMV)
    3.1 Increase resistance to Psg
    3.2 Increased resistance to SMV-GUS
    4. The activated immunity observed in the GmATG2-, GmATG5- and GmATG7-silenced plants do not result from the activated MAPK signaling pathway
    5. Impaired autophagic degradation process in the GmATG2-, GmATG5- and GmATG7- silenced plants
    5.1 The accumulation level of the ATG8 protein enhanced in three differ-ent silenced plants under dark and starvation conditions
    5.2 The different accumulation level of ubiquitinated proteins in three si-lenced plants compare to BPMV-0 plants
    Chapter 4: Discussion
    参考文献
    Appendix
    1. Commonly used medium preparation
    2. Preparation of common reagents and buffers
    3. Primers sequences
    Research Achievements During Study
    致谢
    浙江师范大学学位论文诚信承诺书
    声明
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Silencing Autophagy-Related Genes(ATGs) Results in the Accelerated Senescence and En-hanced Immunity in Soybean

Hashimi Said Masoud
浙江师范大学
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自噬是生物在进化过程中保守的分解代谢途径，在营养循环利用及胁迫适应中起着重要作用。在模式植物拟南芥中对自噬途径已有广泛深入地研究，但在作物中的研究仍十分有限。在此项研究中，我们利用豆荚斑驳病毒(Beanpodmottlevirus,BPMV)介导的基因沉默技术在大豆中沉默了三个在自噬途径的不同阶段发挥关键作用的自噬相关基因(Autophagy-relatedgene,ATG),GmATG2、GmATG5和GmATG7。我们发现这三个基因的沉默植株在正常生长条件下的表型与对照植株并无显著差异；然而在黑暗处理后，这三个基因的沉默植株表现出加速衰老的表型；另外，在黑暗处理后，这三个基因的沉默植株中的水杨酸(Salicylicacid,SA)和ROS(Reactiveoxygenspecies)含量显著增加以及病程相关基因的诱导表达显著增强，说明沉默这三个基因激活了免疫反应；与此免疫激活表型相一致，这三个基因的沉默植株对大豆斑点病菌(Pseudomonassyringaepv.glycinea,Psg)以及大豆花叶病毒(Soybeanmosaicvirus,SMV)的抗性显著增强。以上结果说明自噬在大豆中的功能也是保守的；我们发现GmATG2、GmATG5和GmATG7沉默植株中的免疫激活不依赖于MAPK激酶途径；相反，flg22诱导的GmMPK3/4/6的激活在上述三个沉默植株中反而较对照植株中下降；在黑暗处理条件下ATG8蛋白在三个沉默植株叶片中的积累量显著高于对照植株叶片中的积累量说明三个沉默植株中的自噬降解受损；我们意外发现，与对照株相比，在黑暗处理条件下泛素化蛋白积累水平在GmATG2沉默株中显著升高，而在GmATG5及GmATG7沉默株中则显著降低，说明GmATG2、GmATG5和GmATG7在自噬清除受损的泛素化蛋白中可能发挥着不同作用。综上所述，我们的研究结果表明自噬途径在大豆中负调控免疫反应，该功能在不同植物种中是保守的。

大豆;加速衰老;自噬相关基因;免疫激活

浙江师范大学

硕士

Masters in Biology

Liu Jianzhong

2021

中文

S565.103

2021-11-11（万方平台首次上网日期，不代表论文的发表时间）

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