我国西北部分地区致病疫霉菌无毒基因Avr3α的遗传变异研究
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摘要
Avr3a是致病疫霉菌(Phytophthora infestans (Mont)de Bary)中第一个被克隆的无毒基因,在致病疫霉菌与寄主互作中,其除了具有无毒功能外,还具有重要的毒性功能。本研究的目的是分析我国西北地区致病疫霉菌中Avr3a基因的遗传变异情况,以期更完整地了解无毒蛋白AVR3a的毒性变异机制,同时检测相应抗性基因R3a在这些地区的有效性;对病菌群体进行交配型测定后可检测交配型与Avr3a无毒基因变异的相关性,另外,掌握病菌A2交配型的发生情况,可推测病菌群体有性生殖发生的可能性。
研究中采用对峙培养法对2007和2008年分离自我国西北部分地区的致病疫霉菌进行交配型测定;利用PCR-SSCP技术、DNA测序和Southern杂交方法来检测致病疫霉菌中Avr3a基因的遗传变异情况。
交配型测定结果显示,有A2交配型菌系存在;对140个分离物的PCR-SSCP分析未发现有Avr3a基因核苷酸变异存在,交配型与其变异与否无明显关系;代表分离物的DNA测序结果表明,所有Avr3a基因全部为avr3a基因(相对Avr3a基因而言有毒性的基因);Southern分析结果表明,代表分离物未发生拷贝数差异变化。
综合分析可推测,我国西北部分地区致病疫霉菌Avr3a/avr3a基因变异类型单一,由此说明无毒基因Avr3a的毒性变异机制相对简单;同时在某种程度上可以证明这些地区的致病疫霉菌毒性较强,含R3a抗性基因的马铃薯品种在该地区可能已无抗病效果;在140个供试菌系中没有发现Avr3a基因缺失的菌系,说明该基因对病菌可能具有重要作用;病菌的交配型与该基因的变异没有明显关系;在甘肃、宁夏两省发现了A2交配型菌系,并且分布较为广泛,为病菌有性生殖的发生提供了可能性。
Avr3a was the first cloned avirulence gene in Phytophthora infestans (Mont) de Bary. In the pathogen-host interaction process, avirulence genes play key roles and usually have dual functions, avirulent function which is required for rsistence when being recognized by host plant via cognate R gene and virulent function by manipulating host cell function when the cognate R is absent. The objective of this study was to investigate the genetic diversity of Avr3a gene in the Phytophthora infestans populations in northwestern China, and to aid better understanding of genetic mechanisms involved in virulence variation of avirulence gene Avr3a and to assess the effectiveness of its corresponding resistance gene R3a. Mating type determination allows examination of the relation between mating types and genetic Avr3a variation the possible presence of sexual reproduction.
PCR-SSCP assay, DNA sequencing and Southern blotting were employed to investigate the genetic variations in Avr3a gene in the P. infestans isolates isolated from parts of northwestern China in two consecutive years 2007 and 2008.
Mating type determination identified 21 A2 mating type isolates. The results showed that all 140 isolates examined displayed single PCR-SSCP genotype in both mating types. DNA sequencing of selected isolates further showed that the Avr3a gene were all identical virulent allele avr3a. Southern blotting analysis of selected isolates revealed no copy number variation.
According to the above results, it is apparent that the genetic variation of Avr3a was simple in P. infestans populations in certain areas of northwestern China. The widely presence of simple virulent genotype of avr3a suggests its important role in P. infestans pathogenicity. The results also indicated that the resistance gene R3a in potato is likely ineffective in the areas where the pathogen was sampled. The identification of A2 mating type isolates in Ningxia and Gansu and their wide-range distribution suggests possible presence of sexual reproduction.
研究中采用对峙培养法对2007和2008年分离自我国西北部分地区的致病疫霉菌进行交配型测定;利用PCR-SSCP技术、DNA测序和Southern杂交方法来检测致病疫霉菌中Avr3a基因的遗传变异情况。
交配型测定结果显示,有A2交配型菌系存在;对140个分离物的PCR-SSCP分析未发现有Avr3a基因核苷酸变异存在,交配型与其变异与否无明显关系;代表分离物的DNA测序结果表明,所有Avr3a基因全部为avr3a基因(相对Avr3a基因而言有毒性的基因);Southern分析结果表明,代表分离物未发生拷贝数差异变化。
综合分析可推测,我国西北部分地区致病疫霉菌Avr3a/avr3a基因变异类型单一,由此说明无毒基因Avr3a的毒性变异机制相对简单;同时在某种程度上可以证明这些地区的致病疫霉菌毒性较强,含R3a抗性基因的马铃薯品种在该地区可能已无抗病效果;在140个供试菌系中没有发现Avr3a基因缺失的菌系,说明该基因对病菌可能具有重要作用;病菌的交配型与该基因的变异没有明显关系;在甘肃、宁夏两省发现了A2交配型菌系,并且分布较为广泛,为病菌有性生殖的发生提供了可能性。
Avr3a was the first cloned avirulence gene in Phytophthora infestans (Mont) de Bary. In the pathogen-host interaction process, avirulence genes play key roles and usually have dual functions, avirulent function which is required for rsistence when being recognized by host plant via cognate R gene and virulent function by manipulating host cell function when the cognate R is absent. The objective of this study was to investigate the genetic diversity of Avr3a gene in the Phytophthora infestans populations in northwestern China, and to aid better understanding of genetic mechanisms involved in virulence variation of avirulence gene Avr3a and to assess the effectiveness of its corresponding resistance gene R3a. Mating type determination allows examination of the relation between mating types and genetic Avr3a variation the possible presence of sexual reproduction.
PCR-SSCP assay, DNA sequencing and Southern blotting were employed to investigate the genetic variations in Avr3a gene in the P. infestans isolates isolated from parts of northwestern China in two consecutive years 2007 and 2008.
Mating type determination identified 21 A2 mating type isolates. The results showed that all 140 isolates examined displayed single PCR-SSCP genotype in both mating types. DNA sequencing of selected isolates further showed that the Avr3a gene were all identical virulent allele avr3a. Southern blotting analysis of selected isolates revealed no copy number variation.
According to the above results, it is apparent that the genetic variation of Avr3a was simple in P. infestans populations in certain areas of northwestern China. The widely presence of simple virulent genotype of avr3a suggests its important role in P. infestans pathogenicity. The results also indicated that the resistance gene R3a in potato is likely ineffective in the areas where the pathogen was sampled. The identification of A2 mating type isolates in Ningxia and Gansu and their wide-range distribution suggests possible presence of sexual reproduction.
引文
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