Cloning and Characterization of Pear PLC and S-RNase Genes

The diploid P. x bretschneideri Rehd. is the first pear species with its genome comprehensively sequenced (Wu et al., 2012b (link)). We compared the sequence of our cloned PLC cDNA fragments isolated from pollen tubes with that of the ‘DangshanSuli’ (P. x bretshneideri Rehd.) genome to determine the full-length PLC sequence. The full-length cDNA sequences of S₇-RNase and S₃₄-RNase (DQ414813) were based on the P. x bretschneideri Rehd. genome and GenBank sequences, respectively. The S₇-RNase and S₃₄-RNase cDNA sequences were separately cloned into the pGADT7 vector (Clontech). The PLC cDNA sequence was cloned into the pGBKT7 vector (Clontech). Comparing with the PLC gene mutation of the ‘Jinzhuili,’ we inserted 78 nucleotide residues, which encoded 26 amino acid residues, into the same mutant position in the ‘Dangshansuli’ PLC gene. The mutation of the ‘Dangshansuli’ PLC gene was also cloned into the pGBKT7 vector (Clontech). Each bait/prey pair was introduced to the AH109 yeast strain (Clontech). As a control for auto-activation false-positives, each bait was also co-transformed into the yeast strain with an empty AD vector, and each prey was co-transformed with an empty BD vector. The bait/prey pair colonies that grew on all selective media (Trp-Leu and Trp-Leu-Ade-His) were considered positive for interaction.