Cloning and expression of DNA encoding soybean cytosolic acetyl-CoA carboxylase, soybean fructose-1,6-bisphosphatases, and spinach fructose-1,6-bisphosphatases
Date
1997
Authors
Fuchs, Erica Bates
Major Professor
Advisor
Wurtele, Eve Syrkin
Committee Member
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Abstract
This thesis describes research on the expression of genes encoding soybean cytosolic acetyl-CoA carboxylase (ACCase) isozymes and the chloroplastic and cytosolic fructose- 1,6-bisphosphatases (FBPases) from soybean and spinach. Plant cytosolic ACCase is needed for the production of a number of plant defense compounds, including isoflavonoid antimicrobial phytoalexins. Plant FBPases are needed for production of carbohydrates, which are energy-storing molecules that plants use as food. The goal of the research was to learn about gene expression of cytosolic ACCase and both FBPases during plant defense reactions. The research involved first the cloning of DNA fragments for each enzyme, except
soybean cytosolic FBPase, followed by expression studies.
Work with DNA led to the successful isolation of previously uncloned soybean (cv. Williams 82) cytosolic ACCase-B gene fragments and cDNA fragments encoding the mature forms of chloroplastic FBPase from soybean (cv. Acme) and spinach. The expression studies of soybean cytosolic ACCase and both FBPases looked at MRNA abundance during development and two plant defense reactions, localized plant cell death (the hypersensitive response) and isoflavonoid phytoalexin production, elicited in resistant Rpg4 plants by a P. syringae pv. tomato (avrD) syringolide elicitor.
The results failed to prove that the syringolide elicitor induces cytosolic ACCase mRNA in treated soybean leaves of Rpg4 plants, although mRNAs for the phytoalexin production enzymes PAL and CHS were induced in Rpg4 but not rpg4 soybean plants in a light-independent manner. The syringolide elicitor was not responsible for a transient decrease in the abundance of mRNA encoding chloroplastic and cytosolic FBPase, demonstrating that there is no connection between plant defense reactions and changes in FBPase mRNA levels. The expression studies of spinach chloroplastic FBPase and cytosolic FBPase involved creation of over-expression plasmids by subcloning PCR-amplified cDNA fragments into expression vectors.
Spinach cytosolic FBPase was then produced in large quantities by using as a biofactory a mutant E coli bacterium that lacks its own FBPase. The creation of these over-expression plasmids should allow future investigations on the relationships between enzyme structure and function during plant stress.
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thesis