Vector Construction And Transformation Of The Rice Bentazon And Sulfonylurea Herbicides Resistance Gene Bel

After the complete sequences of the rice genome, post-genome era is coming with the study of the functional genomics. The research of functional plant genomics which focuses on elucidating the function of genes has made rapid progress in recent years. Through Co⁶⁰ radiation rice mutants were obtained which are sensitive to bentazon and sulfonylureas herbidides, after fine mapping, the mutation reason is a single base G deletion at position＋1,332 bp downstream from the translation start codon of CYP81A6. This deletion introduces a premature stop codon and leads to the loss of the heme-binding motif, which is essential for cytochrome P450 function because it contain an absolutely conserved cysteine that serves as the fifth ligand to the heme iron. CYP81A6 presumably functions as a hydroxylase for the detoxification of bentazon and sulfonylureas herbicides in rice named Bel gene.Based on the mapping, the gene Bel is cloned and introduced into Arabidopsis, oilseed rape and tobacco respectively mediated by Agrobacterium tumefaciens strain GV3101. Those transformants confirmed that they were resistant to herbicide bentazon and sulfonylureas. We analyzed the function, segregation law of transformants and plant biological traits of gene Bel. The main results were as follows:1. Extracting the RNA of rice cultivar 'Nipponbare', the Bel gene ORF was obtained by RT-PCR and the length of it is 1542 bp. The Bel gene was cloned to the pGEM-T Easy, then sequenced. The sequence was identical to AK104825 in GenBank.2. We constructed the plant expression vector pCAMBIA3301-BEL based on the pCAMBIA3301, which has a selection marker of bar gene. The Bel fragment replaced the GUS gene of the original plasmid. The expression plasmid was identified by enzyme cutting.3. A total of 32 Arabidopsis transgenic plants were obtained using floral dip method transformation. PCR analysis of the putative transgenic plants revealed that the Bel gene was integrated into the Arabidopsis genome.4. To validate the expression of BeI gene, 4 putative transformation plants of Arabidopsis were identified by RT-PCR. The result indicated this gene expressed in transgenic plants, and the phenotype was not different from wild type plants.5. In order to study the range of tolerant herbicide of Arabidopsis transgenic plants, three kinds of herbicides bentazon, bensulfuron methyl and Basta were tested when Arabidopsis growed about 3 weeks with several concentrations, respectively. The results were as follows: bentazon secure concentration was 0.006%-0.03% of 48% bentazon; bensulfuron methyl secure concentration was 0.1g/L-3g/L; Basta secure concentration was 0.01%-0.6% of 18.5% Basra.6. Oilseed rape (Brassica napus), HUASHUANG 4, was transformed using floral dip method, then 16 putative transgenic plants were obtained, PCR analysis of these palnts showed that the target gene was integrated into the rapeseed genome.7. Tobacco was transformed by tissue culture, then 31 putative transgenic plants were obtained, PCR analysis of these paints showed that the target gene was integrated into the tobacco genome.