稻瘟病菌三个侵染表达上调基因的功能研究
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摘要
稻瘟病菌(Magnaporthe oryzae)是目前研究丝状真菌的重要模式生物。为了探索水稻与稻瘟病菌互作的分子机理,本文从稻瘟病菌全基因组基因芯片中挑选了三个该菌侵染水稻后表达上调倍数比较高的基因,对它们进行基因敲除,同时对其中的两个基因进行过量表达,分析它们的表型变化,以初步确定它们在稻瘟病菌侵染致病过程中的作用。
首先构建了三个基因的敲除载体和MGG_06834.6与MGG_03891.6基因的过量表达载体,然后通过原生质体转化,获得基因的缺失突变体和过量表达突变体。突变体表型分析发现,MGG_03356.6缺失突变体在菌落形态,孢子萌发,致病性方面与野生型没有显著差别,而在疏水性表面上附着胞形成率比野生型菌株KU70低;MGG_06834.6缺失突变体的表型与对照菌株也没有明显变化,但过量表达突变体的产孢量降低,且附着胞形成提前,而致病性减弱;MGG_03891.6的缺失突变体的表型与野生型菌株没有显著差别,但过量表达突变体的附着胞形成滞后,且致病性也有所减弱。RT-PCR分析表明,MGG_06834.6和MGG_03891.6在野生型菌株中表达量很低。这些结果表明它们可能在稻瘟病菌侵染致病过程中充当一定的作用。
同时,我们利用SDS的胁迫来观察细胞壁的完整性,实验结果发现,敲除MGG_03356.6和MGG_03891.6基因对细胞的完整性影响不明显,而敲除MGG_06834.6基因后病菌对SDS抗性较弱,说明细胞完整性减弱,因此MGG_06834.6可能是细胞完整性的调控因子。
Magnaporthe oryzae is one of model organisms in the research of Filamentous fungi. To explore the interaction between Magnaporthe oryzae and its host in molecular level, three genes which high expression during fungal infection were chosen for functional analysis. The knock-out mutans of all three genes and overexpression mutants of two genes were obtained to test their phenotypes in this paper.
The gene deletion mutants of MGG_03356.6, MGG_06834.6 and MGG_03891.6, and gene overexpression mutants of MGG_06834.6 and MGG_03891.6 were obtained. The results indicated that there is no obvious difference between MGG_03356.6 deletion mutant and wild type strain in fungal colony morphology, conidial germination and pathogenicity. However, the appressoria formation on the hydrophobic surface was slightly low in the mutant. The deletion of MGG_06834.6 and MGG_03891.6 genes did not effect the fungus obviously, while the overexpression mutant of MGG_06834.6 showed low conidiation, fast appressoria formation but less virulence, and the overexpression mutant of MGG_03891.6 showed slow appressoria formation, as well as less virulence. The expressions of MGG_06834.6 and MGG_03891.6 in wild type were low by RT-PCR analysis. The results suggestted that these two genes may play certain roles during infection of Magnaporthe oryzae.
Meanwhile, the cell wall integrity of three deletion mutants was tested by SDS stress. The results showed that the mutant of MGG_06834.6 but no MGG_03356.6 and MGG_03891.6 demonstrated weak resistance to the SDS stress, suggesting that the MGG_06834.6 gene may be a regulator in the cell wall integrity.
首先构建了三个基因的敲除载体和MGG_06834.6与MGG_03891.6基因的过量表达载体,然后通过原生质体转化,获得基因的缺失突变体和过量表达突变体。突变体表型分析发现,MGG_03356.6缺失突变体在菌落形态,孢子萌发,致病性方面与野生型没有显著差别,而在疏水性表面上附着胞形成率比野生型菌株KU70低;MGG_06834.6缺失突变体的表型与对照菌株也没有明显变化,但过量表达突变体的产孢量降低,且附着胞形成提前,而致病性减弱;MGG_03891.6的缺失突变体的表型与野生型菌株没有显著差别,但过量表达突变体的附着胞形成滞后,且致病性也有所减弱。RT-PCR分析表明,MGG_06834.6和MGG_03891.6在野生型菌株中表达量很低。这些结果表明它们可能在稻瘟病菌侵染致病过程中充当一定的作用。
同时,我们利用SDS的胁迫来观察细胞壁的完整性,实验结果发现,敲除MGG_03356.6和MGG_03891.6基因对细胞的完整性影响不明显,而敲除MGG_06834.6基因后病菌对SDS抗性较弱,说明细胞完整性减弱,因此MGG_06834.6可能是细胞完整性的调控因子。
Magnaporthe oryzae is one of model organisms in the research of Filamentous fungi. To explore the interaction between Magnaporthe oryzae and its host in molecular level, three genes which high expression during fungal infection were chosen for functional analysis. The knock-out mutans of all three genes and overexpression mutants of two genes were obtained to test their phenotypes in this paper.
The gene deletion mutants of MGG_03356.6, MGG_06834.6 and MGG_03891.6, and gene overexpression mutants of MGG_06834.6 and MGG_03891.6 were obtained. The results indicated that there is no obvious difference between MGG_03356.6 deletion mutant and wild type strain in fungal colony morphology, conidial germination and pathogenicity. However, the appressoria formation on the hydrophobic surface was slightly low in the mutant. The deletion of MGG_06834.6 and MGG_03891.6 genes did not effect the fungus obviously, while the overexpression mutant of MGG_06834.6 showed low conidiation, fast appressoria formation but less virulence, and the overexpression mutant of MGG_03891.6 showed slow appressoria formation, as well as less virulence. The expressions of MGG_06834.6 and MGG_03891.6 in wild type were low by RT-PCR analysis. The results suggestted that these two genes may play certain roles during infection of Magnaporthe oryzae.
Meanwhile, the cell wall integrity of three deletion mutants was tested by SDS stress. The results showed that the mutant of MGG_06834.6 but no MGG_03356.6 and MGG_03891.6 demonstrated weak resistance to the SDS stress, suggesting that the MGG_06834.6 gene may be a regulator in the cell wall integrity.
引文
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[22] Balhadère PV, Talbot NJ. PDEI encodes a P-Type ATPase involved in appressorium-mediated plant infection by the rice blast fungus Magnaporthe grisea[J]. Plant Cell, 2001, 13(9): 1987-2004
[23] Lau GW, Hamer JE. A genetic locus required for conidiophore architecture and pathogenicity in the rice blast fungus[J]. Fungal Genet Biol, 1998, 24(1): 228-239
[24] Liu H, Suresh A, Willard FS, et al. Rgs1 regulates multiple Gαsubunits in Magnaporthe grisea pathogenesis, asexual growth and thigmotropism[J]. EMBO J, 2007, 26: 690-700
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[29] Shi Z, Leung H. Genetic analysis of sporulation of Magnaporthe grisea by chemical and insertional mutagenesis[J]. Mol. Plant Microbe Interact, 1995, 8(5): 949- 959
[30] Shi Z, Leung H. Genetic interactions between spore morphogenetic mutations affect cell types sporulation and pathogenesis in Magnaporthe grisea[J]. Mol. Plant Microbe Interact, 1998, 11(3): 199-207
[31]刘树俊,魏荣宣,有江力,等.稻瘟病菌致病突变体的REMI诱变与鉴定[J].生物工程学报, 1998, 14(3): 254-258
[32]王刚,杨之为,彭友良,等.稻瘟菌产孢和生长基因的鉴定[J].河南大学学报, 2002, 32(3): 11-15
[33]李宏宇,潘初沂,陈涵,等.稻瘟病菌T-DNA插入方法优化及其突变体分析[J].生物工程学报, 2003, 19(4): 419-423
[34] Zhu H, Whitehead DS, Lee YH, et al. Genetic analysis of developmental mutants and rapid chromosome mapping of APP1, a gene required for appressorium formation in Magnaporthe grisea[J]. Mol. Plant Microbe Interact, 1996, 9(9): 767-774
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