水稻WRKY80转录调节蛋白基因的分离与表达模式

doi:10.3864/j.issn.0578-1752.2013.19.009

摘要/Abstract

摘要： 【目的】分离水稻WRKY80，分析其编码蛋白的序列结构特征，了解其在各器官中以及病原接种、激素处理下的表达模式，为阐明其生物学功能奠定基础。【方法】基于水稻基因组数据库注释的Loc_Os03g63810基因的编码序列设计特异引物，用RT-PCR法从茉莉酸甲酯（MeJA）处理的水稻叶片中克隆WRKY80 cDNA 序列，运用生物信息学手段对推定的蛋白和启动子序列进行分析，用Northern杂交或实时荧光定量PCR方法研究基因的表达模式。【结果】获得了WRKY80 cDNA序列，长1 392 bp，包含1个长1 164 bp 的完整开放读码框，编码387个氨基酸。WRKY80具有1个典型的WRKY 保守结构域，锌指类型为C2H2，归类于WRKY 第Ⅱ组；C-端为酸性结构区，且含连续的6个谷氨酰胺和8个丝氨酸，提示具有转录激活活性；预测其定位于细胞核。WRKY80与玉米和高粱等单子叶植物WRKY 的氨基酸序列同一性较高。WRKY80在各器官中组成性表达，在叶、根和穗中表达丰度较高，花中次之，在茎和颖果中丰度较低，且表达具有发育阶段相关性，在成熟叶、根中的表达分别高于幼叶和幼根；受稻瘟病菌、纹枯病菌、MeJA和乙烯利诱导表达，而水杨酸对其表达无影响。其表达模式与启动子顺式元件预测分析的结果基本一致。【结论】WRKY80具有转录因子的结构特征，推测其可能通过茉莉酸/乙烯介导的信号途径参与水稻的防御反应，也可能参与发育调控。

关键词: WRKY转录因子 , 真菌病原 , 基因分离 , 基因表达模式 , 水稻

Abstract: 【Objective】 The objective of this study is to isolate rice WRKY80, to analyze the characteristics of its coding sequence and to investigate its expression patterns in different organs and under pathogen inoculation and hormone application, thus providing a basis for its function identification. 【Method】Specific primer sequences were designed according to the annotated gene Loc_Os03g63810 released in rice genome database. RT-PCR was used to amplify WRKY80 cDNA sequence from RNA pools generated from methyl jasmonate (MeJA)-treated rice leaves. Bioinformatical tools were employed to analyze its deduced protein sequence and cis-elements in its promoter. Northern blot or real-time fluorescence quantification PCR was used to investigate its expression patterns. 【Result】 The obtained cDNA sequence of WRKY80 was 1 392 bp in length, containing an entire open reading frame of 1 164 bp, encoding a polypeptide of 387 amino acid residues consisting of one classic conserved WRKY domain with a zinc finger motif of C2H2, belonging to the WRKY subgroup Ⅱ. WRKY80 possessed an acidic C terminus with consecutive 6 glutamines and 8 threonines, an acidic region possibly responsible for transcription activating activity, and was predicted to be localized in nucleus. WRKY80 shared high identity at the amino acid level with those from monocotyledons Zea mays and Sorghum bicolor. WRKY80 was constitutively expressed in all tested organs. The transcript abundance was relatively higher in leaves, roots and panicles, next was in flowers, and less was in stems and grains. WRKY80 expression was higher in mature leaves and roots than in young leaves and roots, respectively, indicating a developmental stage-related feature. It was rapidly induced by inoculation with Magnaporthe oryzae and Rhizoctonia solani, and also by application of exogenous MeJA and ethephon, whereas salicylic acid exerted no effects on its expression. Consistent with the expression profiles was the prediction result of cis-elements in its promoter. 【Conclusion】WRKY80 possesses structure characteristics as a putative transcription factor. These findings suggest that it may be involved in defense response to the fungal pathogens and also in development regulation by jasmonic acid/ethylene-dependent signaling pathway.

Key words: WRKY transcription factor , fungal pathogen , gene isolation , gene expression pattern , Oryza sativa

彭喜旭, 唐新科, 周平兰, 胡耀军, 邓小波, 王海华. 水稻WRKY80转录调节蛋白基因的分离与表达模式[J]. 中国农业科学, 2013, 46(19): 4035-4043.

PENG Xi-Xu, TANG Xin-Ke, ZHOU Ping-Lan, HU Yao-Jun, DENG Xiao-Bo, WANG Hai-Hua. Isolation and Expression Patterns of Rice WRKY80 Transcription Regulatory Protein Gene[J]. Scientia Agricultura Sinica, 2013, 46(19): 4035-4043.

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