(Prunussalicina)WRKY 33的克隆与表达分析

Cloning and Expression Analysis of PsWRKY 33 Gene from Prunussalicina

WRKY类转录因子是植物防御反应信号转导的重要组成部分,本研究为了探讨WRKY33基因的功能,以嫩叶为材料,采用RT-PCR和RACE技术相结合的方法,克隆得到WRKY33全长c DNA(命名为Ps WRKY33),对其进行生物信息学分析。以基因组DNA为模板,对该基因的ORF区域进行PCR扩增,获得WRKY33的g DNA全长。采用荧光定量PCR技术检测该基因在水杨酸处理下的表达模式。试验结果表明:Ps WRKY33 c DNA全长为2 113 bp,开放阅读框长为1 770 bp,编码589个氨基酸;Ps WRKY33蛋白含有两个WRKY保守结构域,与Pm WRKY33蛋白同源性最高。Ps WRKY33基因组全长为2 844 bp,存在3个内含子和4个外显子。荧光定量结果表明:Ps WRKY33在1 mmol/L水杨酸处理后,30 h表达量最高,呈先上升后下降的趋势。研究结果为深入研究WRKY33基因与水杨酸诱导植物抗病性的关系提供一定的参考价值。

WRKY class transcription factors(TFs) are part of the signal transduction cascade, which leads to the activation of plant defense reactions, In order to further investigate the function of WRKY33. In this experiment,using‘Nai'(Prunussalicina Lindli. var cordata J.Y. Zhang et al.) as experimental materials, the full-length c DNA of WRKY33 gene was cloned by RT-PCR and RACE and named Ps WRKY33. The bioinformatics analysis of WRKY33 was carried out. The Ps WRKY33 genomic DNA has also been amplified. In addition, the expression level of‘Nai'(Prunussalicina) which was treated by 1 mmol/L Salicylic Acid were also analyzed by Real Time PCR. The results indicated that: the full length c DNA of Ps WRKY33 consisted of 2 113 bp base pairs with a1 770 bp open-reading frame encoding 589 amino acids. Systematic cladogram analysis revealed that Ps WRKY33 and Pm WRKY33 from prunusmume could be categorized into the same cluster. The predicted Ps WRKY33 contained two the highly conserved functional domains WRKY. The DNA sequence of the Ps WRKY33 gene was2 844 bp and contained three introns and four exons. The results of Real Time PCR showed that the expression of Ps WRKY33 was increasing first and then decreasing, which had the highest expression at 30 h after salicylic acid challenge treatment. These results might provide a value for the further research about the relation between Ps WRKY33 and SA inducing the plant disease resistance.