用等位基因特异性寡核苷酸(ASO)-PCR快速检测抗多菌灵的油菜菌核病菌  

Rapid Identification of Carbendazim Resistant Strains of Sclerotinia sclerotiorum Using Allele-Specific Oligonucleotide (ASO)-PCR

作　　者：李红霞[1] 周明国[1] 

机构地区：[1]南京农业大学植物保护学院,南京210095

出　　处：《中国农业科学》2004年第9期1396-1399,共4页Scientia Agricultura Sinica

基　　金：国家"863"计划资助项目(2001AA249041)

摘　　要：通过3对兼并性引物扩增获得与S.sclerotiorum抗药性相关的β-微管蛋白基因,全长1 685 bp,包含4个内元,相应的编码447个氨基酸。该基因与其它6种线状真菌的β-微管蛋白基因相比,氨基酸序列同源性达95.78%～97.66%,但内元数目和大小不同。比较S.sclerotiorum敏感型和抗药性菌株的β-微管蛋白基因,发现该基因第198位氨基酸由谷氨酸突变为丙氨酸,从而导致田间抗药性的产生。为了快速、准确监测田间抗药性频率,根据S.sclerotiorumβ-微管蛋白基因的突变位点设计了2对等位基因特异性寡核苷酸(ASO),直接以菌核的基因组DNA为模板扩增,所需时间约为6 h,抗药性检出率为100%,与传统菌丝直径法的测定结果相比检测准确率为96%,而传统的检测方法至少需要1～2周。Benzimidazole fungicides are important mixture components to control rape sclerotinia rot disease in China. Carbendazim-resistant isolates of Sclerotinia sclerotiorum have been detected in fields since 1996. In this paper, three pairs of degenerate primers were used to amplify theβ-tubulin gene from S.sclerotiorum. The gene from S.sclerotiorum has 1 685 bp, including 4 introns, encoding 447 amino acid. Except for the difference in the number of the introns, the deduced amino acid sequence of the β-tubulin gene from S.sclerotiorum are 95.78% to 97.66% identical to those of other six plant pathogenic filamentous fungi. Resistance was related to a point mutation in codon 198 where the glutamic acid has changed into alanine, which caused the occurrence of resistance field. A DNA fragment surrounding codon 198 was amplified directly from genomic DNA of sclerotinia using two pairs of Allele-Specific Oligonucleotide(ASO) primers to detect resistant frequency accurately. Using this method within 6 h, the detection rate for benzimidazole resistance was up to 100%; in comparison with the conventional assay procedure, which needs 1-2 weeks, the accuracy of the new method was 96%.

关 键 词：等位基因 特异性 寡核苷酸(ASO)-PCR检测 抗多菌灵 油菜 菌核病菌 

分 类 号：S435.654[农业科学—农业昆虫与害虫防治]