Abstract:

Here we report the isolation and expression of a pollen-specific cDNA TaPSG719 with an unusually long 5'' leader sequence via suppression subtractive hybridization and 5''/3'' RACE techniques. The insert in the TaPSG719 is 1 172-bp long, and encodes a protein of 188 amino acids long with a pI of 12.1. This sequence did not show a significant homology to any genes deposited in the public database by BLAST search. Southern blot indicated that TaPSG719 might be multicopy. Northern blot and RT-PCR analyses indicated that TaPSG719 transcripts were specific for mature pollen, and undetectable in microspore, immature seed, stem, young leaf, root and ovary. During pollen development, TaPSG719 transcripts were first detectable on the 5th day before anthesis and increased rapidly at middle stages of pollen development with maximum levels on the 4th day before anthesis, and decreased during pollen maturation. It is noted that TaPSG719 contains an unusually long 5'' leader sequence (329-nt) upstream the ATG start codon, suggesting that the gene could be subject to translational regulation. To investigate the role of the 5'' UTR on translation, in vitro transcription/translation assays with various deletion and mutation constructs were performed using wheat germ extract. The results demonstrated that the 5'' UTR affected positively downstream translation in wheat germ extract.

Key words: wheat (Triticum aestivum), pollen-specific cDNA, gene expression, suppression subtractive hybridization