Isolation of the Gene Coding for Movement Protein from Grapevine Fanleaf Virus

Abstract

A pair of degenerate primers, GMPF1 and GMPR1, was designed on the basis of alignment of previously reported Grapevine fanleaf virus (GFLV) movement protein (MP) nucleotide sequences from Iran and other parts of the world. cDNA was synthesized by the use of Oligo d(T)18 from total RNA extraction from each diseased grapevine leaf sample and subjected to polymerase chain reaction (PCR) with the degenerate primers under a range of annealing temperatures from 48 to 62°C. It was revealed that 55°C gave the best result in terms of producing exactly the expected fragment (1044 bp) from as many samples as possible although accompanied by few fade non specific fragments. However, by application of “hot-start" PCR and annealing at 60°C the specific fragment was amplified from 41 out of 86 samples. This was the first amplification of the precise MP cDNA from GFLVs in Iran which is very important as to preparation of recombinant anti-GFLV MP antibody to use in studying the GFLV- grapevine interaction, and also for generating pathogen-derived resistant vines.