Abstract:

【Objective】 Cloning TaPrx gene and analyzing its function preliminarily during interaction between wheat and Puccinia striiformi. 【Method】 A TaPrx gene was cloned by screening cDNA library using PCR combined with RACE, then it was analyzed by bioinformatics. The TaPrx gene was cloned into pET-32a(+) vector, and the recombinant plasmids were transformed into E.coli BL21 (DE3) strain and then was induced by IPTG. The expression pattern of TaPrx gene was analyzed by Real-time RT-PCR. 【Result】 The full length of TaPrx gene was 688 bp and its ORF is 489 bp. It encoded a 162 amino acid protein with calculated molecular weight of 17.36 kD and isoelectric point of 5.32. The deduced protein included one conserved cysteine, however, the signal peptide and transmembrane helices were not found. The prediction of subcellular localization of TaPrx gene was cytoplasmic with 94% probability. The molecular weight of TaPrx fusion protein was 38 kD, and the best induced IPTG concentration was 0.05 mmol/L. The maximum fusion protein was obtained by inducing at 20℃ for 20 h. Real-time RT-PCR indicated that the expression of TaPrx was induced by Puccinia striiformi in wheat, and the highest expression occurred at 24 h and 18 h after inoculation in compatible and incompatible interaction respectively. 【Conclusion】 The polyclonal antiserum of TaPrx gene was obtained. The expression of TaPrx gene was induced by Puccinia striiformis and the TaPrx gene may play a key role in the interaction between wheat and Puccinia striiformi. However, the function of TaPrx gene, which eliminated and regulated ROS in wheat challenged by Puccinia striiformis, needs to be further analyzed.

Key words: wheat, Puccinia striiformis, TaPrx, cloning, prokaryotic expression, Real-time RT-PCR