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乙酰羟酸合酶(AHAS)是磺酰脲类、咪唑啉酮类、三唑嘧啶类、磺酰胺类和嘧啶水杨酸类等乙酰羟酸合酶抑制剂类除草剂的作用靶标,获得能抗此类除草剂的AHAS突变基因资源具有非常重要的理论和应用价值.从长期使用甲磺隆的农田土壤中分离到1株对乙酰羟酸合酶抑制剂类除草剂有广谱抗性的菌株L19,根据表型特征、生理生化特性和16S rDNA序列系统发育分析,将其鉴定为假单孢菌属(Pseudomonas sp.).利用PCR技术从Pseudomonas sp.L19的总DNA中克隆AHAS基因,氨基酸序列比对结果表明,L19与对除草剂敏感菌株P.putida KT2440的AHAS调节亚基氨基酸序列完全相同,而催化亚基有4个氨基酸位点不同:[Val 29→Ala(L19→KT2440),Pro93→Ser,Val 345→Ala,Pro 484→Arg].分别将菌株L19与KT2440的AHAS催化亚基克隆到pET29a(+)的多克隆位点,构建了表达载体pET-L19AHASc和pET-KT2440AHASc,通过镍柱亲和层析纯化得到带有组氨酸标签的乙酰羟酸合酶.抗性试验结果表明菌株L19的乙酰羟酸合酶对四大类乙酰羟酸合酶抑制剂类除草剂的抗性均要强于KT2440的乙酰羟酸合酶,对甲磺隆、咪唑乙烟酸、唑嘧磺草胺和嘧草醚的抗性倍数分别达到53.6、9.3、8.2和9.5倍.菌株L19的乙酰羟酸合酶比活力、对ThDP和Mg2+的Kc值相应地比KT2440乙酰羟酸合酶的要低;而对底物丙酮酸钠的米氏常数Km值要比KT2440乙酰羟酸合酶的要高近1倍.
Acetohydroxy acid synthase (AHAS) is the target of acetohydroxy acid synthase inhibitor herbicides such as sulfonylureas, imidazolinones, triazolopyrimidines, sulfonamides and pyrimidine salicylates, AHAS mutant gene resources resistant to these herbicides have very important theoretical and practical value.A long-term use of metsulfuron-methyl in farmland soil isolated from an acetohydroxy acid synthase inhibitor herbicide broad-spectrum resistance The strain L19 was identified as Pseudomonas sp. According to the phenotypic characteristics, physiological and biochemical characteristics and phylogenetic analysis of 16S rDNA sequence.The AHAS gene was cloned from the total DNA of Pseudomonas sp.L19 by PCR, The results of amino acid sequence alignment showed that L19 had the same amino acid sequence of AHAS regulatory subunit as the herbicide sensitive strain P.putida KT2440, whereas the catalytic subunit had four different amino acid residues: [Val 29 → Ala (L19 → KT2440) , Pro93 → Ser, Val345 → Ala, Pro484 → Arg]. The AHAS catalytic subunits of strains L19 and KT2440 were respectively cloned into the multiple cloning site of pET29a (+), and the expression vectors pET-L19AHASc and pET-KT2440AHASc , Purified by nickel column affinity chromatography with histamine Labeled acetohydroxy acid synthase resistance test results show that the strain L19 acetohydroxy acid synthase four kinds of acetohydroxy acid synthase inhibitor herbicide resistance are stronger than KT2440 acetohydroxy acid synthase, Metsulfuron-methyl, imazethapyr, flumetsulam and pyrithrin were 53.6, 9.3, 8.2 and 9.5 fold, respectively. The specific activity of acetohydroxy acid synthase in strain L19 and the Kc value of ThDP and Mg2 + Which is lower than that of KT2440 acetohydroxy acid synthase; while the Km value of substrate sodium pyruvate is nearly 1 times higher than that of KT2440 acetohydroxy acid synthase.