Studies on Molecular Mechanism of Glyphosate Tolerance in Bean

To reveal molecular basis of glyphosate tolerant bean, the cDN A library of tolerant bean root tip was constructed with λ gt10 as vector. The number of recombinant phages is 4.8×105 .Using two synthetic fragments as pro be, positive clone was obtained by in situ hybridization of λ plaques,and a lso identified by restrict enzyme digestion and Southern hybridization. Sequ ence of cDNA of bean EPSP synthase contains 2 024bp, and the coding region o f 1 569bp codes for a polypeptide of 523 amino acids and one te rmination codon. Comparison of the amino acid sequence homology among many pla nt EPSP synthases indicate that the mature EPSP synthases are highly conserved proteins (more than 84.8%). But there are lower homologous in the chloroplast t ransi t peptide portion. Using the tolerant bean EPSP synthase cDNA sequence informa tion, cDNA fragment of susceptible bean EPSP synthase was amplified by RT-PC R. PCR products cloned into pUC18 and sequenced. The nucleotide sequence ana lysis of cD NAs of the two EPSP synthase revealed a single-point difference, which acco mpanied with one animo acid difference. From these results, it was clarified tha t this single-point difference of nucleotide was the cause of glyphosate-toler ance in the tolerant bean.