Cloning of a Promoter-Like Soybean DNA Sequence Responding to IAA Induction inEscherichia coliK121

摘要

We have constructed a soybean genomic DNA library inEscherichia coliK12 strain KC13 using plasmid pPV33, whichtetracycline resistance(Tcr) gene. A recombinant clone, KC13(pAU-SB1)+, was obtained by selecting for resistance to tetracycline in the presence of indole-3-acetic acid (IAA). Restriction enzyme cleavage and Southern hybridization analysis revealed that the pAU-SB1 plasmid has a 260 bp soybean DNA insert fused with the Tcrgene. In the presence of a selected group of auxins, induction of the Tcrphenotype and mRNA synthesis of the Tcrgene are observed only in KC13(pAU-SB1)+cultures. On the other hand, induction of the Tcrphenotype and mRNA synthesis of the Tcrgene are absent in cells harboring the cloning vector pPV33 or a recombinant plasmid containing the 250 bp insert in the reverse orientation, pAU-SB1ro. This demonstrated a need for the insertion of the 260 bp soybean DNA and the specificity of its orientation in response to IAA induction. The start point of mRNA transcription in response to IAA, IBA, IPA, 2,4,5-T, andα-NAP is at base pair−96 or−95 upstream of the translational start site of the Tcrgene and base pair−98 with