Isolation and Expression Profiles of Cadmium Stress-Responsive Rice WRKY15 Transcription Factor Gene

doi:10.16819/j.1001-7216.2018.7056

Abstract

Abstract:

【Objective】Cadmium (Cd) is one of the most toxic heavy metal elements to growth and development of plants and human health. WRKY transcription factors play important regulatory roles in the resistance against stresses. But so far, the regulatory functions of WRKY proteins (WRKYs) in the resistant responses to Cd stress in plants remain unclear so far. 【Methods】Full-length coding sequence of OsWRKY15 was amplified by RT-PCR with gene-specific primers designed according to gene prediction. Structural characteristics of OsWRKY15 were analyzed by bioinformatics software; Organ-specific and induced expression patterns of OsWRKY15 were analyzed by qRT-PCR. Transcriptional activation activity of OsWRKY15 was tested by yeast one-hybrid assay.【Results】OsWRKY15, 988 bp in length, contained an entire ORF in length of 825 bp, encoding a protein of 274 amino acid residues consisting of one WRKY domain with a zinc finger motif of C₂HC, belonging to the WRKY subgroup III. OsWRKY15 had a nuclear localization signal and was predicted to be localized in nucleus. Yeast one-hybrid assay revealed that OsWRKY15 had transcriptional activation activity. The transcript levels of OsWRKY15 were higher in roots than those in stems and leaves, and the least was in panicles, flowers and dry seeds. Its expression was rapidly and significantly induced in response to Cd exposure, nitric oxide (NO) and abscisic acid (ABA) application with more increase folds in roots than in leaves. However, salicylic acid, jasmonic acid and ethylene exerted no obvious effects on the expression of OsWRKY15. 【Conclusion】OsWRKY15 may be involved in the regulation of rice resistance response to Cd stress through NO- and ABA-dependent signal pathways.

Key words: WRKY transcription factor, cadmium, gene isolation, gene expression profiles, Oryza sativa

摘要：

【目的】镉(Cd)是一种对植物生长发育和人类健康均具有很强毒害效应的重金属元素。WRKY转录因子在抗逆生理过程中起重要的调节作用。为了研究WRKY基因在镉胁迫响应和抗性反应中的作用,【方法】基于基因注释,采用RT-PCR策略从一氧化氮(NO)处理的水稻叶片中分离完整的OsWRKY15编码序列,运用生物信息学软件分析其编码蛋白的结构特征,采用qRT-PCR分析其基因的空间(器官)特异性及诱导表达模式;运用酵母单杂交方法检测其转录激活活性。【结果】克隆了OsWRKY15 cDNA 序列,长988 bp,包含一个长825 bp的完整开放读码框,编码274个氨基酸。OsWRKY15具有1个典型的WRKY保守结构域,锌指类型为C₂HC,归类于WRKY第3组;拥有一个核定位信号,因此推测为一种核蛋白。酵母单杂交分析表明,它具有转录激活活性。OsWRKY15在根中的表达丰度最高,其次是叶和茎,在颖果、花和干种子中的表达丰度较低;受Cd、NO和脱落酸(ABA)快速且显著诱导,且在根中被诱导的程度比叶中大,而水杨酸、茉莉酸和乙烯对其表达无明显影响。【结论】OsWRKY15是一个具有功能的核蛋白,推测其可能通过NO、ABA介导的信号途径参与水稻对Cd胁迫的抗性反应。

关键词: WRKY转录因子, 镉, 基因分离, 基因表达模式, 水稻

CLC Number:

Xixu PENG, Ningning BAI, Haihua WANG. Isolation and Expression Profiles of Cadmium Stress-Responsive Rice WRKY15 Transcription Factor Gene[J]. Chinese Journal OF Rice Science, 2018, 32(2): 103-110.

彭喜旭, 白宁宁, 王海华. 响应镉胁迫的水稻WRKY15转录因子基因的分离与表达特征[J]. 中国水稻科学, 2018, 32(2): 103-110.

Figures/Tables 5

Fig. 1. Schematic diagram of the deduced protein of the OsWRKY15 gene. Dotted, vertically lined, diagonally lined and filled bars represent nuclear localization signal, WRKYGQK peptide, zinc-finger motif and acidic region, respectively. Conserved WRKY domain and zinc-finger pattern are presented, and numbers indicate the amino acid position of each structure in OsWRKY15.

Fig. 2. Phylogenetic tree of OsWRKY15 and WRKY proteins from other species. Bootstrap values from 1000 replicates are shown.

Fig. 3. Expression patterns of OsWRKY15 in different rice organs. Different uppercase letters indicate significant difference at 0.01 level (n=6) according to Duncan’s multiple range test.

Fig. 4. OsWRKY15 expression in response to different treatments with hormones and Cd exposure. A, Cadmium; B, Nitric oxide; C, Salicylic acid; D, Methyl jasmonate; E, Abscisic acid; F, Ethephon; *, ** indicate significant difference at 0.05, 0.01 level(n=6), respectively, in relative to that of the control at 0 h according to Duncan’s multiple range test.

Fig. 5. Assay of transcriptional activation activity of OsWRKY15 in yeast cells. A, Yeast transformants grew in SD-Trp or SD-Trp-Ade-His media. 1, Empty vector pGBKT7 (negative control); 2, pGBKT7-OsWRKY15; 3, pGBKT7-30 (positive control); B, Assay for relative β-galactosidase activity of yeast transformants. Different lowercase letters indicate significant difference at P<0.05 according to Duncan’s multiple range test.

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