大豆疫霉细胞自噬基因PsAtg6a和细胞凋亡相关基因的功能研究
发布时间：2018-08-08 17:52
【摘要】：正常的细胞死亡有助于维持生物体生理活动的运行。生物的细胞死亡包括细胞程序性死亡(programmed cell death,PCD)以及细胞坏死。而细胞程序性死亡又包括细胞凋亡和细胞自噬,其中细胞自噬是细胞内保守的降解过程,这一过程可以将细胞内损伤的细胞器或者废弃的蛋白质等降解,生成的物质重新被细胞利用。在外部环境不利如饥饿条件,或者内部压力如损伤的细胞器的聚集,或者病原菌的侵入时,细胞自噬都能帮助细胞存活,抵抗压力。细胞自噬的发生包括诱导发生、自噬体的形成、自噬体与溶酶体融合以及最终物质的降解等过程。现在研究发现细胞自噬在许多真核生物如丝状真菌、植物、果蝇、线虫、昆虫、人的生理病理方面都发挥着重要的作用。而细胞凋亡是生物体内由基因控制的主动死亡的过程,大量研究表明它在生长发育及致病性等方面发挥着重要功能,但大豆疫霉的细胞凋亡和细胞自噬还知之甚少。为了研究大豆疫霉中是否发生细胞自噬,之前研究发现雷帕霉素可以诱导细胞自噬的发生。通过PEG介导的大豆疫霉的稳定转化获得GFP-PsAtg8的过表达转化子,通过雷帕霉素处理前后GFP-PsAtg8融合蛋白的位置变化来观察细胞自噬的发生。荧光显微镜观察结果表明对照组(DMSO)的菌丝荧光聚集较亮,而雷帕霉素处理后的菌丝中荧光分散,说明菌丝确实发生了细胞自噬。同时通过MDC染色法观察大豆疫霉细胞自噬的发生,它是一种广泛使用的自噬荧光染料。结果显示与对照相比,经雷帕霉素处理的菌丝中自噬体数量增多,荧光亮点增加。这两种方法说明大豆疫霉中可以发生细胞自噬。为了进一步探究细胞自噬是否参与到大豆疫霉的不同生长发育阶段,利用MDC染色的方法观察大豆疫霉不同生长发育阶段细胞自噬的发生,包括菌丝、孢子囊形成、休止胞以及休止胞萌发。结果表明MDC染色结果发现初生孢子囊中MDC荧光强,而成熟的孢子囊的MDC荧光较弱。休止胞以及休止胞萌发阶段MDC染色结果显示休止胞的MDC荧光较弱,而休止胞萌发时MDC荧光增强,说明在孢子囊形成,休止胞萌发时细胞自噬过程被激活。我们由此可以推断细胞自噬可能在大豆疫霉侵染时发挥作用。为了进一步研究细胞自噬在大豆疫霉产胞和致病性方面的作用,在大豆疫霉中鉴定了在侵染阶段持续上调表达细胞自噬相关基因PsAtg6a。通过PEG介导的大豆疫霉原生质体转化的方法获得PsAtg6a沉默转化子,并接种感病品种大豆黄化苗下胚轴。结果显示与野生型P6497相比,PsAtg6a沉默后其致病力降低,用台盼蓝染侵染部位的表皮细胞发现吸器量减少。沉默细胞自噬相关基因PsAtg6a导致孢子囊的产生减少,结合之前MDC染色结果显示的孢子囊形成阶段自噬增强这一结论,表明细胞自噬在孢子囊形成阶段发挥重要的作用。通过上述研究,我们在大豆疫霉中初步证明了细胞自噬的发生,并且发现细胞自噬可能参与大豆疫霉孢子囊产生以及致病性,这些结果将会加深我们对大豆疫霉中细胞自噬对其生长发育和致病性影响的认识。为了研究另一种程序性细胞死亡-细胞凋亡在大豆疫霉侵染过程中可能的作用,在大豆疫霉中鉴定到了四个细胞凋亡相关基因:PsCYCS、PsEndoG和PsAIF,PsTatDs,其中PsTatDs包括PsTatD1,PsTatD2,PsTatD3和PsTatD4。通过荧光定量PCR方法分析这7个基因在大豆疫霉侵染过程中的表达情况。PsAIF在侵染阶段上调表达,PsEndoG在侵染前期有轻微上调表达,PsCYCS在侵染阶段下调表达,PsTatD2和PsTatD3在侵染阶段上调表达,PsTatD4在侵染前期下调表达而在侵染后期上调表达。结果显示PsEndoG,PsCYCS 和 PsAIF,PsTatD2,PsTatD3,PsTatD4 可能在大豆疫霉侵染过程中发挥重要作用。
[Abstract]:Normal cell death contributes to the maintenance of biological activity. Cell death in organisms includes programmed cell death (programmed cell death, PCD) and cell necrosis. Cell programmed death includes cell apoptosis and cell autophagy, in which autophagy is a conservative degradation process in the cell, and this process will be fine. The degradation of intracellular organelles or discarded proteins, resulting in the use of cells. In the external environment, such as hunger conditions, or the accumulation of internal pressure such as damaged organelles, or the invasion of pathogens, cell autophagy can help cells survive and resist stress. The formation of autophagosomes, the fusion of autophagosomes and lysosomes, and the degradation of the final substance. Now studies have found that cell autophagy plays an important role in many eukaryotes, such as filamentous fungi, plants, Drosophila, nematodes, insects, and human beings, and the cell withering is a process of active death in the organism controlled by genes. A large number of studies have shown that it plays an important role in growth, development and pathogenicity, but the cell apoptosis and autophagy of Phytophthora sojae are little known. In order to study the autophagy in Phytophthora soybean, it was found that rapamycin could induce autophagy. The stability of Phytophthora soybean mediated by PEG The overexpression transformant of GFP-PsAtg8 was obtained, and the cell autophagy was observed by the change of the position of GFP-PsAtg8 fusion protein before and after rapamycin treatment. The fluorescence microscope observation showed that the fluorescence aggregation of the hyphae of the control group (DMSO) was brighter, and the fluorescence of the filaments after rapamycin treatment showed that the mycelium did occur. Autophagy was used to observe the autophagy of Soybean Phytophthora cells by MDC staining. It was a widely used autophagy dye. The results showed that the number of autophagic bodies increased and the fluorescent bright spots were increased in the hypha treated by rapamycin. These two methods indicated that autophagy could occur in Phytophthora soybean. Whether autophagy is involved in the different growth and development stages of Phytophthora sojae, MDC staining was used to observe the occurrence of autophagy in the cells of Phytophthora sojae at different growth stages, including mycelium, sporum formation, resting cell and resting cell germination. The results showed that the results of MDC staining showed that the fluorescence of MDC in the primary spores was strong, but the mature spores were found. The MDC fluorescence of the sac was weak. The result of the resting cell and the resting cell germination stage MDC staining showed that the MDC fluorescence of the resting cell was weak, while the MDC fluorescence was enhanced at the time of the resting cell germination, indicating that the process of autophagy was activated when the spore bursa was formed and the resting cell germinated. In order to further study the role of autophagy in the cytoplasm and pathogenicity of Phytophthora sojae, it was identified in Phytophthora soja that the expression of autophagy related gene PsAtg6a. was continuously up-regulated at the infecting stage to obtain PsAtg6a silenced transplants by PEG mediated transformation of Phytophthora soybean, and inoculated the susceptible varieties of soybean yellow seeded seedlings. The results showed that the pathogenicity of PsAtg6a was lower than that of wild type P6497. The epidermal cells of the infected site of trypan blue dye found a decrease in the amount of sucker. The silent cell autophagy related gene PsAtg6a resulted in the decrease of the spores, and the conclusion that the autophagy of the sporospora was enhanced by the result of MDC staining. Autophagy plays an important role in the formation stage of the sporac. Through the above studies, we have demonstrated that cell autophagy in Phytophthora sojae has been preliminarily demonstrated, and that autophagy may participate in the production and pathogenicity of Phytophthora sojae. These results will enhance the growth and development of autophagy in Phytophthora sojae. Understanding of the effect of pathogenicity. In order to study the possible role of another programmed cell death cell apoptosis in the infection process of Phytophthora sojae, four apoptosis related genes were identified in Phytophthora sojae: PsCYCS, PsEndoG and PsAIF, PsTatDs, of which PsTatDs includes PsTatD1, PsTatD2, PsTatD3 and PsTatD4. through fluorescent quantitative PCR methods The expression of these 7 genes in the infection process of Phytophthora sojae.PsAIF is up-regulated at the infecting stage. PsEndoG is slightly up expression at the early stage of infection, PsCYCS is down expression in the infecting stage, PsTatD2 and PsTatD3 are up-regulated at the infecting stage, and PsTatD4 is expressed at the early stage of infection and up-regulated at the later stage of infection. The result shows P. SEndoG, PsCYCS and PsAIF, PsTatD2, PsTatD3 and PsTatD4 may play an important role in the infection process of Phytophthora sojae.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.651
本文编号：2172560
[Abstract]:Normal cell death contributes to the maintenance of biological activity. Cell death in organisms includes programmed cell death (programmed cell death, PCD) and cell necrosis. Cell programmed death includes cell apoptosis and cell autophagy, in which autophagy is a conservative degradation process in the cell, and this process will be fine. The degradation of intracellular organelles or discarded proteins, resulting in the use of cells. In the external environment, such as hunger conditions, or the accumulation of internal pressure such as damaged organelles, or the invasion of pathogens, cell autophagy can help cells survive and resist stress. The formation of autophagosomes, the fusion of autophagosomes and lysosomes, and the degradation of the final substance. Now studies have found that cell autophagy plays an important role in many eukaryotes, such as filamentous fungi, plants, Drosophila, nematodes, insects, and human beings, and the cell withering is a process of active death in the organism controlled by genes. A large number of studies have shown that it plays an important role in growth, development and pathogenicity, but the cell apoptosis and autophagy of Phytophthora sojae are little known. In order to study the autophagy in Phytophthora soybean, it was found that rapamycin could induce autophagy. The stability of Phytophthora soybean mediated by PEG The overexpression transformant of GFP-PsAtg8 was obtained, and the cell autophagy was observed by the change of the position of GFP-PsAtg8 fusion protein before and after rapamycin treatment. The fluorescence microscope observation showed that the fluorescence aggregation of the hyphae of the control group (DMSO) was brighter, and the fluorescence of the filaments after rapamycin treatment showed that the mycelium did occur. Autophagy was used to observe the autophagy of Soybean Phytophthora cells by MDC staining. It was a widely used autophagy dye. The results showed that the number of autophagic bodies increased and the fluorescent bright spots were increased in the hypha treated by rapamycin. These two methods indicated that autophagy could occur in Phytophthora soybean. Whether autophagy is involved in the different growth and development stages of Phytophthora sojae, MDC staining was used to observe the occurrence of autophagy in the cells of Phytophthora sojae at different growth stages, including mycelium, sporum formation, resting cell and resting cell germination. The results showed that the results of MDC staining showed that the fluorescence of MDC in the primary spores was strong, but the mature spores were found. The MDC fluorescence of the sac was weak. The result of the resting cell and the resting cell germination stage MDC staining showed that the MDC fluorescence of the resting cell was weak, while the MDC fluorescence was enhanced at the time of the resting cell germination, indicating that the process of autophagy was activated when the spore bursa was formed and the resting cell germinated. In order to further study the role of autophagy in the cytoplasm and pathogenicity of Phytophthora sojae, it was identified in Phytophthora soja that the expression of autophagy related gene PsAtg6a. was continuously up-regulated at the infecting stage to obtain PsAtg6a silenced transplants by PEG mediated transformation of Phytophthora soybean, and inoculated the susceptible varieties of soybean yellow seeded seedlings. The results showed that the pathogenicity of PsAtg6a was lower than that of wild type P6497. The epidermal cells of the infected site of trypan blue dye found a decrease in the amount of sucker. The silent cell autophagy related gene PsAtg6a resulted in the decrease of the spores, and the conclusion that the autophagy of the sporospora was enhanced by the result of MDC staining. Autophagy plays an important role in the formation stage of the sporac. Through the above studies, we have demonstrated that cell autophagy in Phytophthora sojae has been preliminarily demonstrated, and that autophagy may participate in the production and pathogenicity of Phytophthora sojae. These results will enhance the growth and development of autophagy in Phytophthora sojae. Understanding of the effect of pathogenicity. In order to study the possible role of another programmed cell death cell apoptosis in the infection process of Phytophthora sojae, four apoptosis related genes were identified in Phytophthora sojae: PsCYCS, PsEndoG and PsAIF, PsTatDs, of which PsTatDs includes PsTatD1, PsTatD2, PsTatD3 and PsTatD4. through fluorescent quantitative PCR methods The expression of these 7 genes in the infection process of Phytophthora sojae.PsAIF is up-regulated at the infecting stage. PsEndoG is slightly up expression at the early stage of infection, PsCYCS is down expression in the infecting stage, PsTatD2 and PsTatD3 are up-regulated at the infecting stage, and PsTatD4 is expressed at the early stage of infection and up-regulated at the later stage of infection. The result shows P. SEndoG, PsCYCS and PsAIF, PsTatD2, PsTatD3 and PsTatD4 may play an important role in the infection process of Phytophthora sojae.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.651
【参考文献】
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1 闫思源;姜学军;;细胞自噬及真菌中自噬研究概述[J];菌物学报;2015年05期
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