The use of rolling circle amplification&ndash-RACE (RCA-RACE) for the isolation of full-length genes from peach ( Prunus persica ), involved in fruit development and the study of their role in pit hardening and split-pit process.

摘要

Peach cultivation of clingstone varieties is important for Greek agriculture and canning industry. Split-pits are a recurring problem in peaches (Prunus persica) due to genetic as well as to environmental factors. The occurrence of split-pit formation-the opening of the pit and sometimes splitting of the fruit-causes deterioration of canned fruit quality. To elucidate the molecular mechanisms underlying split-pit formation in peach, we are trying to clone and characterize genes possibly involved, based on the molecular mechanisms underlying fruit dehiscence in the model plant Arabidopsis thaliana. We used both conventional ways as well as a new technique established in our lab called RCA-RACE, which allows the simultaneous isolation of the unknown 3' and 5' ends. PPERFUL and PPERSHP genes that are homologues to the genes FRUITFULL (FUL) and SHATTERPROOF (SHP), respectively, and encode an A- and a C-type MADS-box transcription factors were first isolated. Differences in the mRNA abundance of each gene were compared in a split-pit sensitive and a split-pit resistant variety. Results suggested that temporal regulation of PPERFUL and PPERSHP expression may have an effect on the split-pit process. In the present study, we have cloned and characterized members of CDE-type MADS-box genes, namely PPERAG, PPERSTK, PPERSEP1, PPERSEP3, and PPERFBP9 that are homologous to AGAMOUS (AG), SEEDSTICK (STK), and SEPALLATA (SEP) genes from arabidopsis and FLORAL BINDING PROTEIN 9 (FBP9) from petunia, and try to create a model of genes involved in peach fruit development. Moreover, experiments are under way for the isolation and characterization of genes that act downstream of PPERFUL and PPERSHP, namely PPERALCATRAZ. Finally, peach coding sequences homologous to PHENYLALANINE AMMONIA LYASE, PEROXIDASE, and LACCASE genes that are involved in lignin biosynthesis are analyzed in order to find differences in temporal regulation of lignin formation in split-pit resistant and susceptible varieties.