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Abstract 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 pCambia1300GFP vector, so as to construct the m4AtalsGFP fusion protein overexpression vector with four site mutations.Then the transgenic lines of wild type Arabidopsis thaliana Col0, were obtained by Agrobacterium tumefaciensmediated transformation.The positive transgenic plants were identified by hygromycin resistance screening, and the overexpressed plants were observed by fluorescence microscope and the expression of GFPm4Atals 〖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.
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