Cloning and Characterization of the Meiotic Gene TtRec8 in Tetraploid Wheat

Meiosis is a special cell division that halves chromosomes and generates haploid gametes in eukaryotes. The meiotic cohesin Rec8 has been found to play a significant role in kinetochore orientation and chromosome segregation at meiosis I.  Here, we report the cloning and characterization of the Rec8 homologue in tetraploid wheat (Triticum turgidum L.), designated TtRec8.  The full-length cDNA of TtRec8, encoding 608 amino acids, was cloned from the tetraploid wheat ‘Langdon’ (LDN) through comparative analysis.  Two homoeoloci of TtRec8 were identified on chromosome 1A and 1B, respectively. Real-Time PCR showed significantly higher levels of TtRec8 transcripts in anthers than in roots and leaves.  The expression level of TtRec8 was highest at interphase through early prophase I of meiosis, and then decreased as meiosis proceeded.  Polyclonal antibody against TtRec8 was raised in rabbit using a 471 bp cDNA segment of TtRec8.  Western blotting detected the highest TtRec8 levels in anthers at interphase through early prophase I.  These results indicated that TtRec8 was expressed primarily at interphase through early prophase I as reported with Rec8 in yeast.  TtRec8 antibody was also used to localize endogenous TtRec8 protein in the meiotic cells of anthers at different meiotic stages.  TtRec8 protein was detected along the entire chromosomes at pachytene stage.  Thereafter, most of the TtRec8 protein was removed from the chromosomes.  It was hardly to visualize TtRec8 protein on the chromosomes after pachytene stage.  The kinetics of TtRec8 through the meiotic process in LDN was very similar as the cohesion protein Rec8 in yeast.