稻瘟病菌MoMET3和MoNSR1的基因功能分析

发布时间：2018-05-29 16:45

本文选题：稻瘟病菌 + MoMET3　；参考：《浙江农林大学》2017年硕士论文

【摘要】：由稻瘟病菌(Magnaporthe oryzae)引起的稻瘟病是水稻生产中最为严重的的病害之一。开展稻瘟病菌功能基因的研究,有利于深入认识病菌生物学特性和致病机制,从而为针对性提出防治策略奠定理论基础。同时,稻瘟病菌也是植物病原真菌研究的模式物种之一,可为其它植物病原真菌相关研究提供借鉴。硫元素是生命体生长所必需的元素之一。真菌对硫元素的利用始于ATP硫酸化酶对硫酸根离子的活化。本文对稻瘟病菌ATP硫酸化酶在病菌生长发育和致病过程中的功能进行分析。采用同源重组方法构建基因敲除载体,并对突变体的表型进行分析。结果表明,MoMET3基因是病菌营养生长和分生孢子形成所必需的,但不是毒力因子。稻瘟病菌ATP硫酸化酶编码基因MoMET3缺失后,突变体菌丝生长速度减慢,产孢量降低,但是突变体对寄主的毒力不受影响;虽然基因缺失不影响突变体分生孢子萌发和附着胞成熟,但突变体在硫营养限制时孢子萌发后次生菌丝的生长明显被抑制。MoMET3基因互补后,突变体恢复正常生长,能利用硫酸盐。上述结果表明无机硫的还原对分生孢子的萌发和附着胞的成熟并不是必需的,分生孢子内储存的还原态硫化合物可以满足这一过程中硫营养的需求,但病菌侵入寄主组织后需要吸收利用寄主源还原态硫以利于侵染菌丝的扩展。核仁素在细胞周期及增殖、核糖体的生物合成等过程中发挥重要作用。本文对稻瘟病菌1个核仁素类蛋白MoNSR1在病菌生长发育和致病过程中的功能进行分析。采用同源重组方法构建基因敲除载体,并对突变体的表型进行分析。结果表明,稻瘟病菌MoNSR1基因缺失后,突变体菌丝生长速度减慢,突变体对寄主的毒力下降,但突变体的产孢不受基因缺失的影响。进一步分析,显示基因缺失使得突变体分生孢子的萌发和附着胞的成熟被延迟,细胞壁完整性被破坏,对活性氧、高盐离子、杀菌剂的敏感性增强;对DNA拓扑异构酶I抑制剂的敏感性减弱。上述结果表明Mo NSR1基因在稻瘟病菌的生长发育和致病等过程中均发挥重要作用。
[Abstract]:Rice blast caused by Magnaporthe oryzae is one of the most serious diseases in rice production. The study of functional genes of Pyricularia oryzae is helpful to understand the biological characteristics and pathogenic mechanism of the pathogen and lay a theoretical foundation for the prevention and control strategy. At the same time, rice blast fungus is also one of the model species of plant pathogenic fungi, which can provide reference for other plant pathogenic fungi related research. Sulfur is one of the elements necessary for the growth of life. The use of sulfur by fungi begins with the activation of sulfate ions by ATP sulfatase. In this paper, the function of ATP sulfatase in the growth, development and pathogenicity of rice blast fungus was analyzed. The gene knockout vector was constructed by homologous recombination and the phenotype of the mutant was analyzed. The results showed that MoMET3 gene was necessary for vegetative growth and conidial formation, but not virulence factor. The mycelium growth rate and sporulation of the mutant decreased after MoMET3 deletion, but the virulence of the mutant to the host was not affected, although the gene deletion did not affect the conidial germination and attachment maturation of the mutant. However, the secondary hyphal growth after spore germination was obviously inhibited by the restriction of sulfur nutrition, and the mutants returned to normal growth after complementation of the. MoMET3 gene, and the mutant could utilize sulfate. The results show that the reduction of inorganic sulfur is not necessary for the germination of conidia and the maturation of appressants, and the reduced sulfur compounds stored in conidia can meet the demand of sulfur nutrition in this process. But after invading the host tissues, the bacteria should absorb and utilize the reduced sulfur from the host to facilitate the expansion of the infected hyphae. Nucleolins play an important role in cell cycle and proliferation, ribosomal biosynthesis and other processes. In this paper, the function of a nucleolin-like protein MoNSR1 in the growth and development and pathogenicity of rice blast fungus was analyzed. The gene knockout vector was constructed by homologous recombination and the phenotype of the mutant was analyzed. The results showed that the mycelium growth rate of the mutant decreased after the deletion of MoNSR1 gene and the virulence of the mutant to the host decreased, but the sporulation of the mutant was not affected by the gene deletion. Further analysis showed that gene deletion delayed the germination of conidia and maturation of appressants, destroyed the integrity of cell wall, and enhanced the sensitivity to reactive oxygen species (Ros), high salt ions and fungicides. The sensitivity to DNA topoisomerase I inhibitor was weakened. These results suggest that Mo NSR1 gene plays an important role in the growth, development and pathogenicity of Magnaporthe grisea.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.111.41

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