| 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 |
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| 引用本文： | 李红霞,周明国.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[J].中国农业科学,2004,37(9):1396-1339. |
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| 作者姓名： | 李红霞 周明国 |
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| 作者单位： | 南京农业大学植物保护学院,南京,210095 |
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| 基金项目： | 国家“863”计划资助项目(2001AA249041) |
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| 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。
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| 关 键 词： | 油菜菌核病菌 β-微管蛋白 多菌灵抗药性 等位基因特异性寡核苷酸（ASO）-PCR |
| 收稿时间： | 2003-1-23 |
Rapid Identification of Carbendazim Resistant Strains of Sclerotinia sclerotiorum Using Allele-Specific Oligonucleotide (ASO)-PCR |
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| LI Hong-xia,ZHOU Ming-guo.Rapid Identification of Carbendazim Resistant Strains of Sclerotinia sclerotiorum Using Allele-Specific Oligonucleotide (ASO)-PCR[J].Scientia Agricultura Sinica,2004,37(9):1396-1339. |
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| Authors: | LI Hong-xia ZHOU Ming-guo |
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| Abstract: | 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%. |
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| Keywords: | Sclerotinia sclerotiorum β-tubulin Carbendazim-resistance Allele-Specific Oligonucleotide (ASO-PCR |
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