Towards the Functional Analysis of the AtDOF1.3 Transcription Factor in Tomato

Undergraduate #62
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Courtney Lester - Fort Valley State University
Co-Author(s): Diane Beckles, Ph.D , University of California, Davis, CA

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Tomatoes are one of the most popular vegetables worldwide, due in part to their high levels of healthful lycopene, however, to maintain yields, high levels of nitrogenous fertilizer are needed; this is both expensive, and environmentally damaging. Members of the DNA-binding with One Zinc Finger(zDOF) transcription factor family have been shown to be involved in nitrogen use efficiency (NUE). Transcription factors regulate complex biological processes by controlling the expression of multiple genes that work together in a similar pathway. We wish to functionally characterize AtDOF1.3 to determine if it has a role in controlling NUE in plants. This will be tested by transiently expressing AtDOF1.3 in tomato. The aim of this specific research was to design a cloning strategy to create a recombinant DNA construct for tomato transformation, using a variety of bioinformatics tools. The Open Reading Frame of the AtDOF1.3 was identified using ORFinder in NCBI. Two pairs of nested Polymerase Chain Reaction (PCR) primers were designed using PRIMER3 (i) to amplify AtDOF1.3 and (ii) to create fragments that would permit the gene to be spliced into the multiple cloning site of the pCAMBIA1300 transformation vector. After PCR optimization, a 1.2 kb product of AtDof1.3 was successfully amplified from Arabidopsis thaliana Columbia, genomic DNA using. This PCR product was used to amplify a clonable fragment for ligation into the pCAMBIA after digestion with KpnI and PmeI restriction enzymes, the results of which will be discussed. Agarose gel electrophoresis was performed to confirm that PCR product was of 1.2 kb size.

Not Submitted

Funder Acknowledgement(s): This work was supported by NSF HRD (#1238789) HBCU-UP Targeted Infusion grant to Prof. Seema Dhir at Fort Valley State University, Fort Valley, GA.

Faculty Advisor: Prof. Seema Dhir, dhirs@fvsu.edu

Role: I designed the primers and performed PCR to amplify AtDOF1.3. I then performed gel electrophoresis to confirm that the cloned fragment was of 1.2 kb size.