Cloning auxin-responsive genes for future study in Arabidopsis [abstract]

Abstract

Auxin, a plant hormone, serves as a signal for cells to grow, divide, and differentiate. Approximately 25 years ago, auxin was shown to rapidly affect the expression of specific genes. To date, only three families of auxin responsive genes have been extensively characterized: Aux/IAA, GH3, and SAUR. However, newly published data from another lab has identified other auxin-regulated genes in Arabidopsis thaliana. The purpose of this project was to clone the open reading frames (ORFs) of six of these genes into vectors for over-expression in Arabidopsis. To clone the ORFs of the six genes, primers containing their start and stop codons and restriction sites were designed and used in a reverse transcription polymerase chain reaction (RT-PCR) to amplify the ORFs. RT-PCR was carried out by isolating RNA from Arabidopsis tissue culture and leaf RNA and synthesizing complimentary DNA (cDNA) using oligo dT and reverse transcriptase. The primers were used in a PCR reaction to amplify the ORFs in the cDNA. RT-PCR products were purified and digested at the designed restriction sites. The ORFs were fused to the strong, constituitive 35S CaMV promoter in pUC19, the constructs were transformed into DH5α E. coli cells, and antibiotic resistant colonies were selected. Colonies were screened by PCR using insert-specific primers. Those identified to have the insert by agarose gel electrophoresis were grown in liquid media, so their DNA could be isolated and analyzed by sequencing. The inserts were then cloned into the pPZP211 (an Agrobacterium binary vector), and transformed into DH5α. Cells containing the construct were used in tri-parental mating with Agrobacterium tumefaciens and DH5α RK2013 (containing a helper plasmid). Arabidopsis was vacuum infiltrated using transformed Agrobacterium. Seed of infiltrated plants will be screened to obtain transformed plant lines. Ten to twenty lines will be examined for over-expression of the ORF and any unusual phenotypes. Currently, three of the six constructs have been infiltrated into Arabidopsis. A fourth construct has gone through tri-parental mating, and is in Agrobacterium. The other two constructs are in different stages of the cloning process.