Cloning of tomato SUMO1 and development of a CaMV 35S based gene construct for plant transformation
By: Saad Imran Malik, Abuzar Abdullah, SamraAzam
Key Words: SUMO, Small-ubiquitin-like modifiers, Tomato, CaMV 35S, Over expression.
Int. J. Biosci. 9(5), 86-96, November 2016.Certificate
Tomato (Solanum lycopersicum L.) is one of most important vegetable which is affected by several biotic and abiotic stresses reducing its yield and quality. Stress related proteins may be modified by small ubiquitin-like modifiers (SUMOs) – the process known as SUMOylation, which involves many SUMO proteins and an enzymatic cascade for post-translational modification. SUMOylation is a well-studied process in Arabidopsis but little is known about its roles in crop plants including tomato. This research was aimed to develop aSUMO1 gene overexpression construct under the influence of a CaMV35S promoter. Total RNA was extracted from tomato leaves through Trizol method followed by cDNA synthesis. The SUMO1gene specific primers having BglII and BstEII restriction enzymes sites at 5ʹ ends were used to amplify full-length SUMO1 coding sequence from cDNA via PCR. The fragment was purified and ligated into a TA cloning vector (pGEM-T) followed by sub-cloning in pCAMBIA1301 (a plant transformation vector) from which the native GUS gene was removed. All step-wise confirmations were done by restriction enzyme digestion and colony PCR followed by agarose gel electrophoresis analysis. The resulted plasmid based construct harboring SUMO1 full-length coding sequence was named pCAMBIA: SUMO1. The construct was transformed in Agrobacterium strain LBA4404 through electroporation for subsequent SUMO1 gene transfer in tomato through Agrobacterium-mediated transformation. Thetransgenic plants obtained after transformation will be used for SUMO1 functional studies in tomato regarding biotic/abiotic stress tolerance and disease resistance.