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乙酰羟酸合成酶(AHAS)是磺酰脲类和咪唑啉酮类等AHAS抑制剂类除草剂的作用靶标。获得抗此类除草剂的AHAS突变基因资源具有非常重要的理论和应用价值。本研究从抗甲磺隆菌株Klebsiella sp.HR11和甲磺隆敏感菌株Klebsiella pneumoniae MGH 78578中分别克隆到AHAS三种同工酶基因ilvBN、ilvGM和ilvIH。抗性菌株和敏感菌株AHAS三种同工酶基因在氨基酸水平上差异位点主要集中在ilvBN和ilvGM的大亚基上。将2株菌的ilvBN、ilvGM和ilvIH分别构建到表达载体pET29a(+)中,在Escherichia coli BL21(DE3)中进行表达,测得只有含菌株HR11 ilvBN和ilvGM的转化子细胞破碎液AHAS对各类AHAS抑制剂类除草剂具有较强的抗性,而含菌株HR11 ilvIH和菌株MGH78578 ilvBN、ilvGM和ilvIH的转化子细胞破碎液AHAS对各类AHAS抑制剂类除草剂敏感。
Acetoxyacid synthase (AHAS) is the target of AHAS inhibitor herbicides such as sulfonylureas and imidazolinones. Obtaining AHAS mutant gene resources against these herbicides has very important theoretical and practical value. In this study, three AHAS isoenzymes, ilvBN, ilvGM and ilvIH, were cloned from the isolates of Klebsiella sp. HR11 and the susceptible strain of Klebsiella pneumoniae MGH 78578, respectively. Resistant strains and sensitive strains AHAS three isozyme genes at the amino acid level differences mainly concentrated in the ilvBN and ilvGM large subunit. The two strains of ilvBN, ilvGM and ilvIH were respectively constructed into the expression vector pET29a (+) and expressed in Escherichia coli BL21 (DE3). Only the AHAS of the cell lysate containing the strains HR11 ilvBN and ilvGM AHAS inhibitor herbicide has strong resistance, and strain AH11 inhibitor strains of herbicide HR11 ilvIH and strain MGH78578 ilvBN, ilvGM and ilvIH AHC inhibitor crushed solution AHAS sensitive.