Abstract：Chemical-induced male sterility (CIMS) system, which depends on some chemical hybridization agents (CHAs) such as tribenuron-methyl (TBM) herbicide, is one of the important ways for heterosis utilization in rapeseed (Brassica napus). Selective breeding TMB herbicide resistant rapeseed as male parent in CIMS system can simplify the protocol and cut the cost of hybrid seed production for better yield and quality of rapeseed. Acetohydroxyacid synthase (ALS) is a key enzyme in the biosynthetic pathway of branched-chain amino acids, including leucine, isoleucine and valine. Several herbicidal ALS-inhibitors can bind to ALS and inhibit the activity of ALS, resulting in plant death by blocking the synthesis of branched-chain amino acids. Sulfonylurea (SU) is a kind of herbicidal ALS-inhibitors such as TBM. In this study, we cloned the mutated acetolactate synthase gene (GenBank No. EU520490) from a TBM resistant native flixweed (Descurainia sophia) mutant (#108), and the mutated gene was named as DsALS-108. The results from amino acid sequence alignments revealed a substitution of Pro with Thr at position 197 according to Arabidopsis acetolactate synthase AtALS protein sequence (named P197T). In order to verify whether the P197T mutation in DsALS-108 gene was responsible for the TBM resistance, expression vector PBI121-DsALS-108 was constructed and Agrobacterium tumefaciens carrying the constructs was used for transformation of Arabidopsis. We obtained twelve positive transgenic Arabidopsis lines. When treated with ≥10-4 g/L of TBM, the positive transgenic plants grew normally. In contrast, wild-type plants could not survive. These results suggested that the expression of mutated gene DsALS-108 resulted in the TBM resistance. In Agrobacterium-mediated transformation of rapeseed, hypocotyls were cut as explants. Agrobacterium tumefaciens harboring pCAMBIA3301-DsALS-108 constructs was used to generate transgenic rapeseed plant. Twenty-three positive transgenic lines were obtained. The positive transgenic rapeseed lines expressing DsALS-108 were found to grow well with the treatment of 7.5×10-3 g/L of TBM, which was three times higher than lethal concentration of wild-type plants. Moreover, male fertility of the DsALS-108-expressing rapeseed plants was not affected when treated with by 0.05 mg/L of TBM, which was the concentration used for induction of male sterility, showing the potential application of DsALS-108-expressing rapeseed plants as male parents for hybrid rapeseed production using CIMS. This study can supply new herbicide resistant breeding material for utilization of heterosis in rapeseed using CIMS.
