本文選題：稻瘟病菌 + 過(guò)氧化物酶體��； 參考：《南京農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：稻瘟病是水稻上的三大病害之一,由稻瘟病菌(Magnaporthe oryzae)引起。對(duì)稻瘟病菌的深入研究對(duì)于防治水稻稻瘟病具有重要意義。稻瘟病菌是一種重要的絲狀子囊真菌,并具有典型的致病機(jī)制和侵染循環(huán),其與水稻之間的互作已成為研究病原真菌與植物之間互作的重要模式。隨著研究的深入,發(fā)現(xiàn)過(guò)氧化物酶體對(duì)稻瘟病菌的致病性起著至關(guān)重要的作用。過(guò)氧化物酶體(peroxisome)是真核生物細(xì)胞中普遍存在的一類(lèi)單層膜細(xì)胞器,其內(nèi)含有豐富的酶類(lèi),參與多種生理生化的代謝過(guò)程,如乙醛酸循環(huán)、活性氧的調(diào)節(jié)以及脂肪酸的β-氧化等。過(guò)氧化物酶體由內(nèi)質(zhì)網(wǎng)產(chǎn)生,自身不含DNA,基質(zhì)蛋白和膜蛋白是由核基因編碼,在細(xì)胞質(zhì)中合成,靠過(guò)氧化物酶體定位信號(hào)(Peroxisome targeting signal,PTS)識(shí)別并轉(zhuǎn)運(yùn)到過(guò)氧化物酶體內(nèi)。參與過(guò)氧化物酶體形成的基因稱為PEX基因,編碼蛋白稱為Peroxins。PTS1的受體是PEX5基因,PTS2的受體是PEX7基因,輔助受體是PEX20基因。本研究組曾對(duì)稻瘟病菌PEX7(MoPEX7)和PEX20(MoPE20)進(jìn)行了初步分析,發(fā)現(xiàn)兩個(gè)基因?qū)Σ【L(zhǎng)發(fā)育和致病性均有較大的影響,但具體作用并不完全相同。為了進(jìn)一步探究PEX7和PEX20在過(guò)氧化物酶體形成中的具體分工和在病菌生長(zhǎng)發(fā)育過(guò)程中的作用的異同,本文通過(guò)分析稻瘟病菌MoPEX7和MoPEX20基因的單敲和雙敲突變體,對(duì)稻瘟病菌PEX7、PEX20及PEX7PEX20基因的功能進(jìn)行了比較和分析,結(jié)果如下:1.構(gòu)建含有G418抗性基因(NEO)的MoPEX7基因置換載體,通過(guò)ATMT導(dǎo)入△mopex20(實(shí)驗(yàn)室保存,潮霉素抗性),獲得雙敲突變體△pex20△pex7.2.在CM培養(yǎng)基上,△pex7、△pex20及△pex20△pex7的生長(zhǎng)速率上沒(méi)有明顯差異,△mopex7的菌落形態(tài)較野生型沒(méi)有明顯差異,產(chǎn)孢量有下降;而△mopex20和△pex20△pex7的菌落氣生菌絲明顯變得稀薄,產(chǎn)孢量下降。3.觀察含有PTS1與PTS2的蛋白定位發(fā)現(xiàn),△pex7、△pex20及△pex20△pex7突變體中PTS1的定位與野生型無(wú)差異,PTS2的定位均造成影響。4.測(cè)定在大麥和水稻上的致病性,發(fā)現(xiàn)△pex7、△pex20及△pex20△pex7的致病性均減弱,其中△pex20△pex7的致病性減弱更為顯著。5.利用 MM、MM-C、MM-C+0.5%Tween80、MM-C+0.5%Olive、MM-C+0.5%Oleic acid、MM-C+50mM CH3COONa培養(yǎng)基進(jìn)行營(yíng)養(yǎng)利用試驗(yàn),發(fā)現(xiàn)△pex7、△pex20及△pex20△pex7均不能正常利用長(zhǎng)鏈脂肪酸,但可以利用CH3COONa做碳源,但利用率稍有下降。6.在含有 100g/ml Congo red 和 150g/ml calcofluor white 的培養(yǎng)基上,△pex7、△pex20及△pex20pex7突變體的生長(zhǎng)均受到抑制。在Congo red培養(yǎng)基上△pex7抑制率與△pex20相差不大,在calcofluor white培養(yǎng)基上△?ex7的抑制率與△pex20相差不明顯,而△pex20△pex7的抑制率明顯大。說(shuō)明這兩個(gè)基因是相互作用共同調(diào)節(jié)細(xì)胞壁的完整性。7.在含有Methylviologen的培養(yǎng)基上培養(yǎng),發(fā)現(xiàn)△pex7、△pex20及△pex20△pex7突變體對(duì)活性氧的耐受性與野生型相比下降,同時(shí)△pex20和△pex20△pex7的耐受性較△pex7為低.8.在Terylene膜上對(duì)誘導(dǎo)孢子萌發(fā)和附著胞的形成,發(fā)現(xiàn)△pex7、△pex20及△pex20△pex7突變體的萌發(fā)率與野生型相比沒(méi)有明顯差異,△pex7附著胞形成率與△pex20相差不大,而△pex20△pex7的附著胞形成率明顯降低.9.觀察△pex7、△pex20及△pex20△pex7突變體在大麥葉片上的侵染結(jié)構(gòu)發(fā)現(xiàn),在接種36h后,突變體都能成功侵染并形成侵染菌絲,但△pex7的侵染菌絲比△pex20的少。10.對(duì)△pex7、△pex20及△pex20△pex7突變體的黑色素產(chǎn)量測(cè)定發(fā)現(xiàn),△pex7的黑色素產(chǎn)量明顯高于△pex20和△pex20△pex7.11.對(duì)突變體進(jìn)行脂肪粒染色發(fā)現(xiàn),附著胞形成過(guò)程中△pex7、△pex20及△pex20△pex7孢子和芽管內(nèi)殘存有脂肪粒,并且始終不能轉(zhuǎn)移入附著胞中。綜上所述,突變體△辦ex7和△pex20在菌落形態(tài)、產(chǎn)孢量、致病性、細(xì)胞壁完整性、黑色素產(chǎn)量及對(duì)活性氧的耐受性等方面均存在不同程度的差異。暗示著,在過(guò)氧化物酶體形成過(guò)程中,除了作為PTS2共受體與P一起參與蛋白轉(zhuǎn)運(yùn)之外,PEX-20可能還參與別的途徑。
[Abstract]:Rice blast is one of the three major diseases on rice, which is caused by Magnaporthe oryzae. The deep study of blast fungus is of great significance to the prevention and control of rice blast. The rice blast fungus is an important filamentous sac fungus, and has a typical pathogenic mechanism and invasion cycle. The interaction between rice blast and rice has become a study. An important mode of interaction between pathogenic fungi and plants. With the development of research, peroxisomes have been found to play a vital role in the pathogenicity of blast fungus. Peroxisome (peroxisome) is a kind of monolayer cell organelle commonly found in eukaryotic cells. It contains abundant enzymes and participates in a variety of physiological and biochemical processes. Metabolic processes, such as the glyoxylic acid cycle, the regulation of reactive oxygen species and the beta oxidation of fatty acids. Peroxisomes are produced by the endoplasmic reticulum, themselves without DNA, the matrix protein and membrane proteins are encoded by nuclear genes, synthesized in the cytoplasm, and are identified and transported to peroxisomes by the peroxisome positioning signal (Peroxisome targeting signal, PTS). In the enzyme, the gene involved in peroxisome formation is called the PEX gene, the receptor called Peroxins.PTS1 is the PEX5 gene, the PTS2 receptor is the PEX7 gene and the auxiliary receptor is the PEX20 gene. The study group has carried out a preliminary analysis on the PEX7 (MoPEX7) and PEX20 (MoPE20) of rice blast fungus, and found that two genes were developed and developed for the pathogen. In order to further explore the similarities and differences between PEX7 and PEX20 in the specific division of peroxisome formation and the role of PEX20 in the growth and development of the pathogen, this paper analyzes the single knockout and double knockout mutants of MoPEX7 and MoPEX20 genes of rice blast fungus, and PEX7, PEX20 of rice blast fungus, in order to further explore the difference between the specific roles of the peroxisome formation and the growth and development of the peroxisome. The function of PEX7PEX20 gene was compared and analyzed. The results are as follows: 1. construct a MoPEX7 gene replacement carrier containing G418 resistance gene (NEO), and import Delta mopex20 (laboratory preservation, hygromycin resistance) through ATMT, and obtain the growth rate of delta pex20 Delta pex7.2. on CM pericpero, Delta Pex7, Delta pex20 and delta pex20 Delta. There was no obvious difference between the colony morphology of delta mopex7 and the decrease of the sporulation, while the colony of delta mopex20 and delta pex20 Delta Pex7 became thinner and the sporulation decreased.3., which contained the protein location of PTS1 and PTS2, and the location of PTS1 and the wild type in the delta Pex7, Delta pex20 and delta pex20 Delta Pex7 mutant. The pathogenicity of.4. in barley and rice was influenced by the localization of PTS2, and the pathogenicity of delta Pex7, Delta pex20 and delta pex20 Delta Pex7 were all weakened, and the virulence of delta pex20 Delta Pex7 was weakened more significantly by MM, MM-C, MM-C+0.5%Tween80, and MM-C+0.5%Tween80. It was found that delta Pex7, Delta pex20 and delta pex20 Delta Pex7 could not use long chain fatty acids normally, but CH3COONa could be used as carbon source, but the utilization ratio of.6. was slightly decreased on the medium containing 100g/ml Congo red and 150g/ml Calcofluor white, and the growth of delta and delta mutant were suppressed. The inhibitory rate of delta Pex7 on the NGO red medium was not quite different from Delta pex20. The inhibition rate of delta EX7 on the Calcofluor white medium was not significantly different from Delta pex20, while the inhibition rate of delta pex20 Pex7 was significant. It showed that the two genes were interacted together to regulate the cell wall integrality and were cultured on the medium containing Methylviologen. The tolerance of delta Pex7, Delta pex20 and delta pex20 Delta Pex7 was lower than that of wild type, while the tolerance of delta pex20 and delta pex20 Delta Pex7 was lower than Delta Pex7 as low.8. in Terylene membrane, which showed the formation of spore germination and attachments on Terylene membrane, and found Delta Pex7. There was obvious difference in the formation rate of delta Pex7 attachments and delta pex20, while the formation rate of delta pex20 Delta Pex7 obviously decreased.9. observation Delta Pex7, Delta pex20 and delta pex20 Delta Pex7 mutant on the wheat leaf infection structure found that after inoculation 36h, the mutant could successfully infect and form infecting mycelium, but Delta Pex7 infecting mycelium The determination of melanin yield of delta Pex7, Delta pex20 and delta pex20 Delta Pex7 mutant less than Delta pex20 found that the yield of delta Pex7 was significantly higher than Delta pex20 and delta pex20 Delta pex7.11. for the mutation of the mutant, Delta Pex7, Delta pex20, Delta pex20 and delta spores and the residual fat particles in the bud tube. In summary, the mutant Delta EX7 and delta pex20 have different degrees of difference in colony morphology, sporulation, pathogenicity, cell wall integrity, melanin yield and tolerance to reactive oxygen species, suggesting that in the process of enzyme formation of peroxisomes, except as PTS2 co receptor and P, Apart from protein transport, PEX-20 may also be involved in other pathways.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S435.111.41

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