Transgene detection and papaya ringspot virus coat protein expression in genetically modified Philippine solo papaya (Carica papaya L.)

The transgene was detected in genetically modified Philippine papaya and the expression of the papaya ringspot virus (PRSV) coat protein (CP) was analyzed in three selected T3 events. Southern blot analysis using DNA probe targeting the transgene sequence from the right border to part of the CP gene and Asel-digested genomic DNA from T3 generations of events 124, 132, and 142 showed the presence of the expected approx. 4.1 kb transgene insert. The transcripts of the transgene were detected by RT-PCR. Amplified cDNA of the 318 bp and 464 bp gene fragments of CP and nptII respectively were obtained from all three events, as well as the full length transcript of 792 bp nptII gene but but the transcript of the 935 bp CP gene was not detected. A non radioactive protocol for northern blot analysis was developed but is not sensitive enough for the quantification of gene expression at the transcript level. The expression of the transgene at the protein level was detected by western blot analysis using PRSV antiserum and anti-nptII. Both proteins present in all three events were smaller than the positive controls. The level of transgene-expressed PRSV CP was highest in plants from event 132 as determined by ELISA. The amount of nptII in transgenic plants does not vary greatly between plants of the same event and among the three events analyzed. The mean values obtained by quantitative ELISA were 840, 824, and 842 pg nptII/g leaf tissue in events 124, 132, and 142, respectively. The possible sequence of the expressed coat protein was derived from the previously cloned sequence of 318 bp fragment of the CP gene. Analysis of the protein sequence based on the criteria proposed by FAO and WHO showed no homology to allergenic proteins and absence of an 8-contiguous amino acid motif identical to allergenic proteins in the SDAP indicating no allergenic potential of the novel protein in transgenic Philippine Solo papaya.