Transformation into Arabidopsis

1. Characterization of a Gene that Responds to Mitochondrial Retrograde Regulation in Arabidopsis thaliana Jennifer Sepulveda and David M. Rhoads, PhD. School of Plant Sciences, The University of Arizona, 85721 Undergraduate Biology Research Program, Western Alliance for the Expansion of Student Opportunities, and The Ronald E. McNair Achievement Program • Transformation into Arabidopsis • Successfully inserted the correct construct into Agrobacterium in a reverse orientation. • Floral dip transformation • Kanamycin resistance selection • Left: Mature Arabidopsis plants ideal for floral dipping. Right: T1 Arabidopsis from GV3101Col 0. transformation • Future Plans • Identify homozygous mutants with an At5g40690 insertion at a single locus • Quantify gene expression using Northern Blots • Analyze over-expressor and knockout lines using fungal infection and other environmental stresses • Acknowledgments • Undergraduate Biology Research Program: HHMI Grant #52005889 • Western Alliance for the Expansion of Student Opportunities (NSF) • Ronald E. McNair Achievement Program, Program Director: Dr. Maria Teresa Velez Constructing Constitutive Expressor (CE) Lines for At5g40690 At5g40690 will be characterized through analysis of the overexpression and absence of the gene. The construct for constitutive expression of At5g40690 was created through a cloning experiment and inserted into Arabidopsis through floral dips using Agrobacterium tumefaciens. We believe that plants with constitutive expression of At5g40690 will have increased resistance to pathogen attack and other environmental stresses. Introduction The Rhoads Lab found an Arabidopsis gene (At5g40690) that encodes for a protein similar to yeast ATPase Assembly Proteins (AAPs), which is strongly increased in expression by MRR and during plant stresses such as pathogen attack. Figure of ATPase: AAPs are involved in the assembly of ATPases, which convert ATP to ADP, Pi and energy. Characterization of At5g40690 will be performed by the analysis of knock out (KO) lines, constitutive expressor (CE) lines in comparison to wild-type using northern blots. If this gene is an AAP, then this will be the first AAP in plants to be extensively studied in relation to MRR and stress responses, and will provide researchers with a better understanding of MRR and define a new category of proteins involved in stress response. Findings will give a better understanding of heat stress in crops such as Zea mays . Purify plasmid with At5g40690 Digest with NcoI and BamHI, Purify EcoRI ADP+Pi 5613bp pUNI51: At5g40690 3188bp 673bp pBSKS(+):35S: HSP23.6:T 5915bp 645bp NotI PstI BamHI PstI Amplify gene PstI NcoI ATP pBSKS(+):35S At5g40690 BamHI NcoI PstI PstI 2H+ Ligation and Transformation PstI Digest with PstI, Purify 2H+ 4128bp 1784bp pBSKS(+):35S: At5g40690:T 5912bp pPZP211 9014bp PstI PstI BamHI PstI NcoI Digest with PstI and Dephosphorylation, Purify NcoI BamHI PstI PstI Ligation and Transformation Verification of Correct Construct for Transformation into Agrobacterium 1: Molecular weight marker 2: HindIII digest shows NcoIBamHI orientation 3: HindIII digest shows BamHINcoI orientation 5: EcoRV digest shows NcoIBamHI orientation 6: EcoRV digest shows BamHINcoI orientation 8&9: PstI digest verifies both contain At5g40690 insert (1756bp) HindIII PstI EcoRV NcoI 1 2 3 4 5 6 7 8 9 HindIII BamHI pPZP211:At5g40690 10774bp PstI BamHI EcoRV NcoI