Cloning of PCR-Amplified Coat Protein Gene of Tomato Yellow Leaf Curl Virus

Abstract

Coat protein (CP) gene of tomato yellow leaf curl virus (TYLCV), Thai isolate, was amplified from the DNA extract of plant tissues using polymerase chain reaction (PCR). Two chains of oligonucleotides corresponding to nucleic acid sequences of 5’ and 3’ ends of CP gene of TYLCV were used as primers. Amplification of CP gene was performed for 30 cycles and the products were determined by gel electrophoresis. Approximately 25ug of CP gene-DNA was obtained from the amount of 100-500 pg of viral template existed in DNA extract. The PCR-CP gene was further cloned into SmaI cut pUC 19, and transformed into E. Coli. Clones with CP gene inserted were selected for the construction of a chimeric gene for plant transformation.