大豆疫霉細(xì)胞自噬基因PsAtg6a和細(xì)胞凋亡相關(guān)基因的功能研究
發(fā)布時間：2018-08-08 17:52
【摘要】：正常的細(xì)胞死亡有助于維持生物體生理活動的運(yùn)行。生物的細(xì)胞死亡包括細(xì)胞程序性死亡(programmed cell death,PCD)以及細(xì)胞壞死。而細(xì)胞程序性死亡又包括細(xì)胞凋亡和細(xì)胞自噬,其中細(xì)胞自噬是細(xì)胞內(nèi)保守的降解過程,這一過程可以將細(xì)胞內(nèi)損傷的細(xì)胞器或者廢棄的蛋白質(zhì)等降解,生成的物質(zhì)重新被細(xì)胞利用。在外部環(huán)境不利如饑餓條件,或者內(nèi)部壓力如損傷的細(xì)胞器的聚集,或者病原菌的侵入時,細(xì)胞自噬都能幫助細(xì)胞存活,抵抗壓力。細(xì)胞自噬的發(fā)生包括誘導(dǎo)發(fā)生、自噬體的形成、自噬體與溶酶體融合以及最終物質(zhì)的降解等過程�，F(xiàn)在研究發(fā)現(xiàn)細(xì)胞自噬在許多真核生物如絲狀真菌、植物、果蠅、線蟲、昆蟲、人的生理病理方面都發(fā)揮著重要的作用。而細(xì)胞凋亡是生物體內(nèi)由基因控制的主動死亡的過程,大量研究表明它在生長發(fā)育及致病性等方面發(fā)揮著重要功能,但大豆疫霉的細(xì)胞凋亡和細(xì)胞自噬還知之甚少。為了研究大豆疫霉中是否發(fā)生細(xì)胞自噬,之前研究發(fā)現(xiàn)雷帕霉素可以誘導(dǎo)細(xì)胞自噬的發(fā)生。通過PEG介導(dǎo)的大豆疫霉的穩(wěn)定轉(zhuǎn)化獲得GFP-PsAtg8的過表達(dá)轉(zhuǎn)化子,通過雷帕霉素處理前后GFP-PsAtg8融合蛋白的位置變化來觀察細(xì)胞自噬的發(fā)生。熒光顯微鏡觀察結(jié)果表明對照組(DMSO)的菌絲熒光聚集較亮,而雷帕霉素處理后的菌絲中熒光分散,說明菌絲確實發(fā)生了細(xì)胞自噬。同時通過MDC染色法觀察大豆疫霉細(xì)胞自噬的發(fā)生,它是一種廣泛使用的自噬熒光染料。結(jié)果顯示與對照相比,經(jīng)雷帕霉素處理的菌絲中自噬體數(shù)量增多,熒光亮點增加。這兩種方法說明大豆疫霉中可以發(fā)生細(xì)胞自噬。為了進(jìn)一步探究細(xì)胞自噬是否參與到大豆疫霉的不同生長發(fā)育階段,利用MDC染色的方法觀察大豆疫霉不同生長發(fā)育階段細(xì)胞自噬的發(fā)生,包括菌絲、孢子囊形成、休止胞以及休止胞萌發(fā)。結(jié)果表明MDC染色結(jié)果發(fā)現(xiàn)初生孢子囊中MDC熒光強(qiáng),而成熟的孢子囊的MDC熒光較弱。休止胞以及休止胞萌發(fā)階段MDC染色結(jié)果顯示休止胞的MDC熒光較弱,而休止胞萌發(fā)時MDC熒光增強(qiáng),說明在孢子囊形成,休止胞萌發(fā)時細(xì)胞自噬過程被激活。我們由此可以推斷細(xì)胞自噬可能在大豆疫霉侵染時發(fā)揮作用。為了進(jìn)一步研究細(xì)胞自噬在大豆疫霉產(chǎn)胞和致病性方面的作用,在大豆疫霉中鑒定了在侵染階段持續(xù)上調(diào)表達(dá)細(xì)胞自噬相關(guān)基因PsAtg6a。通過PEG介導(dǎo)的大豆疫霉原生質(zhì)體轉(zhuǎn)化的方法獲得PsAtg6a沉默轉(zhuǎn)化子,并接種感病品種大豆黃化苗下胚軸。結(jié)果顯示與野生型P6497相比,PsAtg6a沉默后其致病力降低,用臺盼藍(lán)染侵染部位的表皮細(xì)胞發(fā)現(xiàn)吸器量減少。沉默細(xì)胞自噬相關(guān)基因PsAtg6a導(dǎo)致孢子囊的產(chǎn)生減少,結(jié)合之前MDC染色結(jié)果顯示的孢子囊形成階段自噬增強(qiáng)這一結(jié)論,表明細(xì)胞自噬在孢子囊形成階段發(fā)揮重要的作用。通過上述研究,我們在大豆疫霉中初步證明了細(xì)胞自噬的發(fā)生,并且發(fā)現(xiàn)細(xì)胞自噬可能參與大豆疫霉孢子囊產(chǎn)生以及致病性,這些結(jié)果將會加深我們對大豆疫霉中細(xì)胞自噬對其生長發(fā)育和致病性影響的認(rèn)識。為了研究另一種程序性細(xì)胞死亡-細(xì)胞凋亡在大豆疫霉侵染過程中可能的作用,在大豆疫霉中鑒定到了四個細(xì)胞凋亡相關(guān)基因:PsCYCS、PsEndoG和PsAIF,PsTatDs,其中PsTatDs包括PsTatD1,PsTatD2,PsTatD3和PsTatD4。通過熒光定量PCR方法分析這7個基因在大豆疫霉侵染過程中的表達(dá)情況。PsAIF在侵染階段上調(diào)表達(dá),PsEndoG在侵染前期有輕微上調(diào)表達(dá),PsCYCS在侵染階段下調(diào)表達(dá),PsTatD2和PsTatD3在侵染階段上調(diào)表達(dá),PsTatD4在侵染前期下調(diào)表達(dá)而在侵染后期上調(diào)表達(dá)。結(jié)果顯示PsEndoG,PsCYCS 和 PsAIF,PsTatD2,PsTatD3,PsTatD4 可能在大豆疫霉侵染過程中發(fā)揮重要作用。
[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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S435.651
本文編號：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.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S435.651
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 閆思源;姜學(xué)軍;;細(xì)胞自噬及真菌中自噬研究概述[J];菌物學(xué)報;2015年05期
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