JrGRAS2 转录因子参与 Dof 转录调控和 HSP 蛋白表达，对高温胁迫耐受有贡献。
The walnut transcription factor JrGRAS2 contributes to high temperature stress tolerance involving in Dof transcriptional regulation and HSP protein expression.
机构信息
Laboratory of Walnut Research Center, College of Forestry, Northwest A & F University, Yangling, 712100, Shaanxi, China.
Key Laboratory of Economic Plant Resources Development and Utilization in Shaanxi Province, College of Forestry, Northwest A & F University, Yangling, 712100, Shaanxi, China.
出版信息
BMC Plant Biol. 2018 Dec 20;18(1):367. doi: 10.1186/s12870-018-1568-y.
BACKGROUND
GRAS transcription factor (TF) family is unique and numerous in higher plants with diverse functions that involving in plant growth and development processes, such as gibberellin (GA) signal transduction, root development, root nodule formation, and mycorrhiza formation. Walnut tree is exposed to various environmental stimulus that causing concern about its resistance mechanism. In order to understand the molecular mechanism of walnut to adversity response, a GRAS TF (JrGRAS2) was cloned and characterized from Juglans regia in this study.
RESULTS
A 1500 bp promoter fragment of JrGRAS2 was identified from the genome of J. regia, in which the cis-elements were screened. This JrGRAS2 promoter displayed expression activity that was enhanced significantly by high temperature (HT) stress. Yeast one-hybrid assay, transient expression and chromatin immunoprecipitation (Chip)-PCR analysis revealed that JrDof3 could specifically bind to the DOFCOREZM motif and share similar expression patterns with JrGRAS2 under HT stress. The transcription of JrGRAS2 was induced by HT stress and up-regulated to 6.73-11.96-fold in the leaf and 2.53-4.50-fold in the root to control, respectively. JrGRAS2 was overexpressed in Arabidopsis, three lines with much high expression level of JrGRAS2 (S3, S7, and S8) were selected for HT stress tolerance analysis. Compared to the wild type (WT) Arabidopsis, S3, S7, and S8 exhibited enhanced seed germination rate, fresh weight accumulation, and activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) and glutathione-S-transferase (GST) under HT stress. In contrast, the Evans blue staining, electrolyte leakage (EL) rates, hydrogen dioxide (HO) and malondialdehyde (MDA) content of transgenic seedlings were all lower than those of WT exposed to HT stress. Furthermore, the expression of heat shock proteins (HSPs) in S3, S7, and S8 was significant higher than those in WT plants. The similar results were obtained in JrGRAS2 transient overexpression walnut lines under normal and HT stress conditions.
CONCLUSIONS
Our results suggested that JrDof3 TF contributes to improve the HT stress response of JrGRAS2, which could effectively control the expression of HSPs to enhance HT stress tolerance. JrGRAS2 is an useful candidate gene for heat response in plant molecular breeding.
背景
GRAS 转录因子（TF）家族在高等植物中是独特而多样的，具有多种功能，涉及植物生长和发育过程，如赤霉素（GA）信号转导、根发育、根瘤形成和菌根形成。核桃树暴露于各种环境刺激物中，这引起了人们对其抗性机制的关注。为了了解核桃对逆境的分子机制，本研究从核桃中克隆并表征了一个 GRAS TF（JrGRAS2）。
结果
从 J. regia 基因组中鉴定出 JrGRAS2 的 1500bp 启动子片段，其中筛选了顺式元件。这个 JrGRAS2 启动子显示出表达活性，高温（HT）胁迫显著增强。酵母单杂交试验、瞬时表达和染色质免疫沉淀（Chip）-PCR 分析表明，JrDof3 可以特异性地结合 DOFCOREZM 基序，并在 HT 胁迫下与 JrGRAS2 具有相似的表达模式。JrGRAS2 的转录受 HT 胁迫诱导，在叶片中上调至对照的 6.73-11.96 倍，在根中上调至对照的 2.53-4.50 倍。JrGRAS2 在拟南芥中过表达，选择了三个 JrGRAS2 高表达水平的株系（S3、S7 和 S8）进行 HT 胁迫耐受性分析。与野生型（WT）拟南芥相比，S3、S7 和 S8 在 HT 胁迫下表现出更高的种子萌发率、鲜重积累、过氧化氢酶（CAT）、过氧化物酶（POD）、超氧化物歧化酶（SOD）和谷胱甘肽-S-转移酶（GST）活性。相比之下，转基因幼苗的 Evans 蓝染色、电解质渗漏（EL）率、过氧化氢（HO）和丙二醛（MDA）含量均低于 HT 胁迫下的 WT。此外，S3、S7 和 S8 中的热休克蛋白（HSPs）表达水平明显高于 WT 植物。在正常和 HT 胁迫条件下，JrGRAS2 瞬时过表达核桃株系也得到了类似的结果。
结论
我们的结果表明，JrDof3 TF 有助于提高 JrGRAS2 对 HT 胁迫的反应，从而有效控制 HSPs 的表达，增强 HT 胁迫耐受性。JrGRAS2 是植物分子育种中耐热反应的一个有用候选基因。
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