【作者】 詹儒林； 黄俊生；

【Author】 Zhan Rulin~(1,2*) Huang Junsheng~1 1.State Key Laboratory of Tropical Crop Biotechnology,Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,Chine;2.Southern Subtropical Crops Research Institute,Chinese Academy of Tropical Agricultural Seienee,Zhanjiang 524091,China

【机构】 中国热带农业科学院热带作物生物技术国家重点实验室、热带生物技术研究所；

【摘要】 芒果炭疽病是我国南方芒果的重要病害。我们在芒果园中分离筛选到了炭疽病菌对多菌灵高抗的菌株,并从该菌株中克隆了β-微管蛋白基因,将其序列与野生型菌株的β-微管蛋白基因进行比对。结果表明,抗性菌株的β-微管蛋白氨基酸在第181、198、237和363等位点均发生了改变,对这些位点用等位特异PCR 的方法在抗性菌株和野生型菌株中分别进行验证,发现只有第198位的突变,用阳性引物进行扩增时,在抗性菌株中扩增到了特异片段,而在野生型菌株中则没有。而用阴性引物进行扩增时,只有在野生型菌株中扩增到特异片段,在抗性菌株中则没有。因此初步证明了β-微管蛋白第198位氨基酸的突变是引起抗药性的主要原因。为了对198位的抗性突变作进一步的验证,我们发现,由于该位点的突变,在第197位和198位间形成了一个酶切位点ACCⅡ(CGCG),因此扩增一段较为保守的含第198位氨基酸的329bp 片段,并用ACCⅡ进行酶切,结果表明,在抗性菌株可以中把329bp 的片段酶切成107bp 和222bp 的两段,而在野生型菌株中则没有这个现象。因此进一步验证了β-微管蛋白第198位氨基酸的突变是引起芒果炭疽病菌对多菌灵形成抗药性的主要原因。更多还原

【Abstract】 Mango anthracnose caused by Colletotrichum gloeosporioides is an important disease prevalent intropical regions of China.High carbendazim(MBC)-resistant field strains were tested and collected.The fragmentsof tub2 were cloned,sequenced,and alignments were carried out between MBC-resistant and wild-type strains ofC.gloeosporioides.The results showed that the amino acids were altered in codons 181 198 237 and 363.All ofthe mutant positions were detected by allele-specific PCR.The results showed that the expected phenomenon dis-appeared exclusively at codon 198,the allele-specific fragments of which were amplified in MBC-resistant strainsby the positive primers but not in wild-type strains.On the contrary,the allele-Specific fragments were amplifiedin wild-type strains by the negative primers but not in MBC-resistant strains.The preliminary findings proved thatthe point mutation occurred at amino acid codon 198,causing a change from glutamic acid(GAG)to alanine(GCG),which is closely associated with confering MBC-resistance in the field.An enzyme assay was employed tofurther test the above results.It involved the creation ofa ACC Ⅱ restriction site(CGCG)at codons 197 and 198(GACGAG→GACGCG)in MBC-resistant strains,in which ACC Ⅱ digested a 329bp fragement(containing code198 and no other ACC Ⅱrestriction sites)into 107bp and 222bp,while the fragments from wild-type strains re-mained undigested.Based on the above assays,all of the MBC-resistant and wild-type strains were detected suc-cessfully.It strongly suggested that the altered at amino acid codon 198 played the leading role in conferringMBC-resistance in Mango anthracnose in south China.更多还原