以Ypt1基因为靶标的致病疫霉菌Phytophthora infestans的分子检测

Molecular detection of Phytophthora infestans based on Ypt1 gene

三磷酸鸟苷(GTP)结合蛋白基因(Ypt1)是一个与原癌基因Ras(Rat sarcoma)相关的基因,在酵母中,该基因编码一个与Ras相关GTP结合蛋白。为了研究以Ypt1基因为分子靶标的致病疫霉菌(P.infestans)检测技术,比较了30种卵菌Ypt1基因的序列,以该序列为靶标设计了1对针对P.infestans的特异性PCR引物Pi1/Pi2。试验结果表明,在供试的55种不同疫霉菌和真菌的144个菌株中,利用这1对引物只能从P.infestans基因组DNA中分别扩增出1条分子量为369 bp的特异性条带,这1对引物的检测灵敏度为100 pg。以疫霉菌Ypt1通用引物Yph1F/Yph2R结合这1对特异引物进行套式PCR扩增,使引物Pi1/Pi2的检测灵敏度提高了10倍,检测到10 pg的基因组DNA。通过套式PCR,引物Pi1/Pi2对游动孢子和卵孢子的检测灵敏度分别为3个游动孢子和1个卵孢子;以Pi1/Pi2引物,分别采用单轮PCR和套式PCR可检测马铃薯发病组织和病田土壤的致病疫霉。以上结果证明Ypt1基因适合作为疫霉菌分子检测靶标。采用以这1对特异引物建立的以PCR技术为基础的分子检测方法,可对田间土壤和发病植物组织中的P.infestans进行快速、灵敏的检测。

Ypt1 is a Ras-related gene,in yeast,the gene encoding a protein associated with GTP binding.In this study,over 30 oomycetes Ypt1 DNA sequences were compared and a pair of primers（Pi1/Pi2） was designed for detecting P.infestans.144 isolates representing 26 species of Phytophthora and 29 fungal species of plant pathogens were used to test the specificity of the primers.PCR amplification with species-specific（Pi） primers resulted in a product of 369 bp only from isolates of P.infestans.The detection sensitivity with Pi primers was 100 pg of genomic DNA.Using Ypt1F/Ypt1R as first-round amplification primers,followed by a second round using the primer pair Pi1/Pi2,a nested PCR procedure was developed,which increased the detection sensitivity 10-fold to 10 pg.For primers Pi1/Pi2,one oospore or 3 zoospores could be detected by nested PCR.The results showed that Ypt1 gene could be a molecular target for detection of P.infestans.PCR with the Pi primers could also be used to detect P.infestans from naturally infected potato tissues and diseased soil samples.