巨桉EgrWRKY70基因克隆和初步表达分析

Cloning and Expression Analysis of Egr WRKY70 in Eucalyptus grandis

WRKY家族为植物所特有,参与调控植物逆境胁迫、生长发育和衰老等生物学过程。为了获得WRKY70同源基因在桉树响应胁迫中的作用和功能,克隆巨桉(Eucalyptus grandis)中的Egr WRKY70基因,通过生物信息学分析和q RT-PCR进行功能的初步鉴定。结果表明,Egr WRKY70基因编码321个氨基酸,蛋白分子量为36.18 ku,只有一个WRKY结构域,属于WRKY转录因子III a类成员。同时,Egr WRKY70与麻风树的Jcu WRKY70的亲缘关系最近,其氨基酸序列相似性达到58.2%。半定量RT-PCR分析表明,Egr WRKY70主要在巨桉叶片、根和花中表达,而在茎部表达量较低。实时定量q RT-PCR分析显示:Egr WRKY70基因在低温和盐胁迫下,表达量快速升高然后下降;在盐胁迫下Egr WRKY70基因表达仍然维持在较高水平;在油菜素内酯和水杨酸处理时,Egr WRKY70基因能够快速的响应,使用茉莉酸甲酯处理时其表达量没有发生明显的变化。由此表明,Egr WRKY70能够通过不同的响应方式参与桉树生物和非生物胁迫的应答。

The plant-specific WRKY transcription factor family appears to be involved in the regulation of various biological processes including response to stresses, growth and development, and senescence. In order to identify the function response to stress of WRKY70 in Eucalyptus grandis, Egr WRKY70 was cloned and analyzed by using reverse transcription polymerase chain reaction（RT-PCR） and real time quantitative PCR（q RT-PCR）. Egr WRKY70 encoded a predict protein of 321 amino acids. The theoretical molecular weight of Egr WRKY70 was 36.18 ku with p I of 5.28, unstability index of 63.67. Egr WRKY70 contained a conserved WRKY domain and belonged to group IIIa. The results of multiple alignments and phylogenetic analysis revealed that Egr WRKY70 closed to Jcu WRKY70 with 58.2% similarity. The expression of Egr WRKY70 was detected in flowers, leaves and roots by semi-quantitative reverse transcription and polymerase chain reaction, and Egr WRKY70 did not nearly expressed in stem including xylem and phloem. Expression of Egr WRKY70 increased firstly and then decreased with the duration of the treatment of salt and cold. However, the Egr WRKY70 expression maintained at a high level under salt stress. Besides, after the treatments of salicylic acid and brassinolide, the levels of Egr WRKY70 expression increased rapidly and then decreased. The expression of Egr WRKY70 almost did not changed after the treatment of methyl jasmonate. These results suggested that Egr WRKY70 may have relationship with biotic and abiotic stress responses in E. grandis.