稻瘟病菌MoPEX7与MoPEX20基因的双敲除及突变体表型分析

发布时间：2018-06-30 19:01

本文选题：稻瘟病菌 + 过氧化物酶体　；参考：《南京农业大学》2016年硕士论文

【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。对稻瘟病菌的深入研究对于防治水稻稻瘟病具有重要意义。稻瘟病菌是一种重要的丝状子囊真菌,并具有典型的致病机制和侵染循环,其与水稻之间的互作已成为研究病原真菌与植物之间互作的重要模式。随着研究的深入,发现过氧化物酶体对稻瘟病菌的致病性起着至关重要的作用。过氧化物酶体(peroxisome)是真核生物细胞中普遍存在的一类单层膜细胞器,其内含有丰富的酶类,参与多种生理生化的代谢过程,如乙醛酸循环、活性氧的调节以及脂肪酸的β-氧化等。过氧化物酶体由内质网产生,自身不含DNA,基质蛋白和膜蛋白是由核基因编码,在细胞质中合成,靠过氧化物酶体定位信号(Peroxisome targeting signal,PTS)识别并转运到过氧化物酶体内。参与过氧化物酶体形成的基因称为PEX基因,编码蛋白称为Peroxins。PTS1的受体是PEX5基因,PTS2的受体是PEX7基因,辅助受体是PEX20基因。本研究组曾对稻瘟病菌PEX7(MoPEX7)和PEX20(MoPE20)进行了初步分析,发现两个基因对病菌生长发育和致病性均有较大的影响,但具体作用并不完全相同。为了进一步探究PEX7和PEX20在过氧化物酶体形成中的具体分工和在病菌生长发育过程中的作用的异同,本文通过分析稻瘟病菌MoPEX7和MoPEX20基因的单敲和双敲突变体,对稻瘟病菌PEX7、PEX20及PEX7PEX20基因的功能进行了比较和分析,结果如下:1.构建含有G418抗性基因(NEO)的MoPEX7基因置换载体,通过ATMT导入△mopex20(实验室保存,潮霉素抗性),获得双敲突变体△pex20△pex7.2.在CM培养基上,△pex7、△pex20及△pex20△pex7的生长速率上没有明显差异,△mopex7的菌落形态较野生型没有明显差异,产孢量有下降;而△mopex20和△pex20△pex7的菌落气生菌丝明显变得稀薄,产孢量下降。3.观察含有PTS1与PTS2的蛋白定位发现,△pex7、△pex20及△pex20△pex7突变体中PTS1的定位与野生型无差异,PTS2的定位均造成影响。4.测定在大麦和水稻上的致病性,发现△pex7、△pex20及△pex20△pex7的致病性均减弱,其中△pex20△pex7的致病性减弱更为显著。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培养基进行营养利用试验,发现△pex7、△pex20及△pex20△pex7均不能正常利用长链脂肪酸,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 的培养基上,△pex7、△pex20及△pex20pex7突变体的生长均受到抑制。在Congo red培养基上△pex7抑制率与△pex20相差不大,在calcofluor white培养基上△?ex7的抑制率与△pex20相差不明显,而△pex20△pex7的抑制率明显大。说明这两个基因是相互作用共同调节细胞壁的完整性。7.在含有Methylviologen的培养基上培养,发现△pex7、△pex20及△pex20△pex7突变体对活性氧的耐受性与野生型相比下降,同时△pex20和△pex20△pex7的耐受性较△pex7为低.8.在Terylene膜上对诱导孢子萌发和附着胞的形成,发现△pex7、△pex20及△pex20△pex7突变体的萌发率与野生型相比没有明显差异,△pex7附着胞形成率与△pex20相差不大,而△pex20△pex7的附着胞形成率明显降低.9.观察△pex7、△pex20及△pex20△pex7突变体在大麦叶片上的侵染结构发现,在接种36h后,突变体都能成功侵染并形成侵染菌丝,但△pex7的侵染菌丝比△pex20的少。10.对△pex7、△pex20及△pex20△pex7突变体的黑色素产量测定发现,△pex7的黑色素产量明显高于△pex20和△pex20△pex7.11.对突变体进行脂肪粒染色发现,附着胞形成过程中△pex7、△pex20及△pex20△pex7孢子和芽管内残存有脂肪粒,并且始终不能转移入附着胞中。综上所述,突变体△办ex7和△pex20在菌落形态、产孢量、致病性、细胞壁完整性、黑色素产量及对活性氧的耐受性等方面均存在不同程度的差异。暗示着,在过氧化物酶体形成过程中,除了作为PTS2共受体与P一起参与蛋白转运之外,PEX-20可能还参与别的途径。
[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.
【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】：S435.111.41

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