Pseudomonas sp.L19抗除草剂AHAS基因的克隆、表达及特性

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.

Cloning,Expression and Characteristics of the Acetohydroxyacid Synthase(AHAS) Gene from Pseudomonas sp.L19

Acetohydroxyacid synthase(AHAS) is the target of AHAS-inhibitor herbicides,such as sulfonylurea,imidazolinone,triazolopyrimidine sulfonamide and pyrimidinyl oxybenzoate.It was recognized that AHAS herbicide-resistant mutant has potential application in the construction of transgenetic herbicide-resistant crop and the development of new potent herbicidal compounds.In this study,an AHAS inhibitor-resistant bacterium L19 was isolated from farm soil treated with metsulfuron-methyl for many years.The strain was preliminarily identified as Pseudomonas sp.according to its morphological,physiological and biochemical properties and the homology analysis of its 16S rDNA sequence.The gene coding for acetohydroxyacid synthase was cloned from Pseudomonas sp.L19 by PCR technique.The AHAS regulatory subunit of Pseudomonas sp.L19 was identical to that of P.putida KT2440,whereas four different sites were found in the catalytic subunit: Val 29→Ala(L19→KT2440),Pro93→Ser,Val 345→Ala,Pro 484→Arg].The AHAS of Pseudomonas sp.L19 and P.putida KT2440 were over-expressed,purified and characterized.The AHAS of Pseudomonas sp.L19 showed multiple resistance to four types of AHAS-inhibitor herbicides.The resistance factors of rAHAS were 53.6-fold,9.3-fold,8.2-fold and 9.5-fold to the AHAS of P.putida KT2440 when metsulfuron-methyl,imazethapyr,flumetsulam and pyriminobac-methyl used as inhibitor,respectively.The specific activity,Kc for ThDP and Mg2+ of Pseudomonas sp.L19 AHAS were lower than that of P.putida KT2440 AHAS,while the Km value of Pseudomonas sp.L19 AHAS for pyruvate was approximately 1-fold higher than the corresponding value of P.putida KT2440.