Plant & Animal Genome IX Conference Plant & Animal Genome IX Conference
Poster: Large Insert Libraries, Gene Isolation, Etc.
P02_14.html
In an attempt to establish the methodology of differential display in our laboratory we compared the gene expression patterns of green Rio Red grapefruit with fruits from the initial stage of ripening, but that had the red color fully developed. We used RNAimage (GeneHunter) as recommended by the manufacturer, without any previous optimization in an attempt to evaluate the potential of the method under sub-optimal conditions. Total RNA was isolated from green and mature Rio Red grapefruit and reverse transcribed using three one base oligo dT anchored primer. PCR was performed using eight 13 mer random primers in combination with the oligo dT anchored primers. The PCR 33P labeled products were separated in 6% polyacrylamide sequencing gel and the X-ray film exposed for 16 h. The differential expressed bands were excised from the gel, reamplified, and cloned. The true positives were identified by reverse northern blot. Twenty four bands displayed specifically in mature fruits, from these, only two were true positives and expressed only in mature fruit as confirmed by northern blot. After sequencing, the two bands shown to be the same fragment, with different sizes, one of them with 330 nucleotides. The comparison of the nucleotide sequence in the GenBank revealed no similarity with other genes, however, it revealed 67% similarity at the amino acid level with Terpene synthase from Citrus junus. The full length cDNA is being obtained by 5' RACE. Considering that it took less than four months to isolate a gene specific for mature grapefruit and that the method is inexpensive, we consider that it payed off by itself, despite of the very high percentage of false positive. We optimized the method afterwards.