油菜乙酰乳酸合成酶抑制剂类除草剂抗性突变体M9的遗传和基因克隆

Inheritance and Gene Cloning of an ALS Inhabiting HerbicideResistant Mutant Line M9 in Brassica napus

【目的】分离和鉴定自然突变的油菜乙酰乳酸合成酶抑制剂类除草剂抗性突变体M9抗性基因,阐明抗性产生的分子基础。【方法】通过配置杂交组合研究其遗传规律,应用同源序列法克隆油菜ALS家族基因,采用杂交转育和小孢子培养技术将抗性基因转育到陆奥-五十铃细胞质雄性不育(MICMS)恢复系中,进行PCR鉴定。【结果】该突变体抗性由1个显性核基因控制。克隆获得BnALS1—BnALS3,核酸序列比对发现,M9抗性是由BnALS1的点突变使蛋白序列的第638位丝氨酸(AGT)残基被天冬酰胺酸(AAT)替代所致,将此抗性基因命名为BnALS1R。抗性基因BnALS1R转育到MICMS恢复系中的PCR检测表明,所有抗除草剂的恢复系都携带有BnALS1R。【结论】突变体M9抗性由1个显性核基因控制,BnALS1第638位丝氨酸突变成天冬酰胺酸是抗性产生的分子基础。

【Objective】The objective of this study is to identify and characterize the resistant gene of a mutant line M9,which was previously found in the rapeseed（brassica napus L.） by spontaneous mutation,conferring resistance to acetolactate synthase or acetohydroxyacid synthase（ALS or AHAS） inhibiting herbicides,and understand the molecular basis for resistance to ALS-inhibiting herbicides.【Method】Reciprocal crosses were made in order to study the inheritance of resistance.Three genes BnALS1-BnALS3 encoding ALS were isolated from the mutant and wild type by using the homology-based candidate gene method.The resistant gene of M9 was transformed into Mutsu-Isuzu cytoplasmic male sterile（MICMS） restorer lines by hybridization,microspore culture,and PCR analysis.【Result】The resistance of M9 was inherited as a single,dominant nuclear gene.Three genes BnALS1-BnALS3 encoding ALS were isolated.Molecular analysis identified a single-point mutation leading to an amino acid substitution from serine 638（AGT） to asparagine（AAT） at the herbicide-binding site of the rapeseed BnALS1 gene.All the resistant restoring lines had the specific band of BnALS1R by sequencing.【Conclusion】The resistance trait of M9 is controlled by a single dominant nuclear gene.The molecular basis for the resistance to ALS-inhibiting herbicides results from the point mutation（Ser638Asn）.