Construction of Sedoheptulose-1,7-Bisphosphatase (Sbpase) for Manipulation in Guard Cells of Arabidopsis thaliana L.

Abstract

Guard cells control the stomata through which exchange of gas takes place in plants by balancing between CO2 uptake for photosynthesis and water loss through transpiration leading to ultimate plant water use efficiency (WUE). Due to climate change, sustainable agriculture will therefore require a major reduction in plant water use hence stomata have become potential target for manipulation. Understanding the signal mechanisms of stomata is therefore critically important in facilitating an understanding of stomatal regulation. The use of molecular tools and techniques to manipulate chloroplast metabolism specifically in the guard cells are needed to elucidate signals associated with stomatal behaviour towards crop improvement. Ability to assemble multiple or complex DNA molecules containing large number of genetic elements is an essential part of genetic engineering and in order to understand the involvement of guard cell photosynthesis in stomatal function, genetic manipulation of photosynthetic enzymes specifically in guard cells is necessary. This study employed the manipulation and construction of the enzyme Sedoheptulose-1,7-Bisphosphatase (SBpase) by using the golden gate cloning technique and the bioinformatics system- geneious. Constructs were designed to alter expression of the SBPase gene in a cell specific manner driven by the guard cell promoter KST1 in the model plant Arabidopsis thaliana L. The construct design for the sense plasmid vectors allowed efficient assembly of multiple DNA fragments in a single reaction based on the type IIs restriction enzyme. The potentials of manipulating guard cell specific metabolism are therefore enormous and the increase or decrease of photosynthetic genes could be assessed and their impacts on plant development documented accordingly.