小麦过氧化物还原酶基因TaPrx的克隆与功能初步分析

Cloning and Primary Characteration Analysis of Peroxiredoxin Gene (TaPrx) from Wheat

【目的】克隆TaPrx基因并对其在小麦与条锈菌互作中的功能进行初步分析。【方法】利用PCR方法结合RACE技术在cDNA文库中筛选得到TaPrx基因的全长序列并进行生物信息学分析,然后克隆至pET-32a(+),转化E.coliBL21(DE3)后用IPTG进行诱导表达。通过Real-timeRT-PCR进行表达模式分析。【结果】得到TaPrx基因的全长序列688bp,ORF489bp,编码162个氨基酸残基,分子量17.36kD,等电点5.32,含一个保守的半胱氨酸残基(Cys),不含信号肽及跨膜结构域,亚细胞定位94%的可能性在细胞质。TaPrx融合蛋白分子量38kD,最佳IPTG诱导浓度0.05mmol·L-1,20℃诱导20h可得到最大量的融合蛋白。Real-timeRT-PCR分析表明TaPrx基因在小麦与条锈菌的亲和与非亲和互作中均受诱导表达,分别在接种后24h、18h达到表达高峰。【结论】获得了TaPrx基因特异性的多克隆抗体;TaPrx基因受条锈菌诱导表达,可能参与了小麦与条锈菌互作。但是否参与了小麦受条锈菌侵染后产生的ROS的清除与调节仍需进一步验证。

【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.