乙酰乳酸合成酶(ALS)是支链氨基酸、缬氨酸、亮氨酸和异亮氨酸生物合成途径中的关键酶，也是多种除草剂的靶点。为了研究als基因不同突变位点组合后其抗除草剂抗性的变化，并整合和增强植株对不同类型除草剂的抗性，本研究对已知抗性位点进行组合并进行了拟南芥转基因分析。我们通过重叠延伸PCR技术体外突变扩增四个已知位点突变的P197S/R199A/W574S/S653F拟南芥Atals，克隆到pCAMBIA1 300GFP载体上，从而构建了四个位点突变的m4AtalsGFP融合蛋白过表达载体。然后用农杆菌介导法转化野生型拟南芥Col0，获得转基因株系。采用潮霉素抗性筛选鉴定阳性转基因植株，并利用荧光体式显微镜观察过表达植株以及在蛋白水平检测GFPm4Atals融合蛋白表达情况。对纯合转基因株系进行除草剂抗性分析。分析表明转基因拟南芥具有磺酰脲和咪唑啉酮两种除草剂的整合抗性。此研究有助于系统地分析als基因不同突变位点对抑制剂的抗性，有效避免和应对自然界als单一位点突变的杂草的困扰。

Acetolactate synthase (ALS) is a key enzyme in the biosynthesis pathway of branched chain amino acids, valine, leucine and isoleucine, and it is also the target of many herbicides.In order to study the changes of herbicide resistance after the combination of different mutation sites of als gene, and to integrate and enhance the resistance of plants to different types of herbicides, four mutated Atals genes with known resistance sites were cloned and transferred into Arabidopsis thaliana.In this research, P197S/R199A/W574S/S653F of Atals with four known site mutations were amplified by overlapping extended PCR in vitro, and cloned into the pCambia1300GFP vector, so as to construct the m4AtalsGFP fusion protein overexpression vector with four site mutations.Then the transgenic lines of wild type Arabidopsis thaliana Col0, were obtained by Agrobacterium tumefaciensmediated transformation.The positive transgenic plants were identified by hygromycin resistance screening, and the overexpressed plants were observed by fluorescence microscope and the expression of GFPm4Atals 〖JP〗fusion protein was detected at the protein level.The herbicide resistance of homozygous transgenic lines was analyzed.The analysis showed that transgenic Arabidopsis thaliana had the integrated resistance of sulfonylurea and imidazolinone.This study is helpful to systematically analyze the resistance of different mutation sites of als gene to inhibitors and effectively avoid and deal with the disturbance of weeds with single site mutation of als in nature.