谷子WRKY36转录因子的分子特性及功能鉴定

doi:10.3864/j.issn.0578-1752.2015.05.03

摘要/Abstract

摘要： 【目的】干旱等非生物胁迫严重影响了植物的生长和作物的产量。WRKY转录因子广泛参与了植物生长发育、形态建成和代谢调控等过程，在调控非生物胁迫响应中也扮演着十分重要的角色。分析谷子SiWRKY36的分子特性和功能，解析谷子转录因子的抗逆调控机制。【方法】通过对干旱胁迫谷子转录组测序结果分析，获得了一个WRKY转录因子SiWRKY36；利用生物信息学的方法分析谷子SiWRKY36的分子特性；根据SiWRKY36蛋白序列进行同源性搜索，得到与谷子SiWRKY36蛋白序列相似度较高的其他物种的蛋白序列；使用MEGA5对谷子SiWRKY36蛋白序列及其同源序列进行多序列比对分析并构建同源物种间系统进化树；利用MEME和SMART在线工具进行蛋白序列分析；利用GSDS和PHYRE2在线工具分别对谷子SiWRKY36基因结构和三级结构进行分析；从谷子基因组数据库Phytozome获取谷子SiWRKY36上游2 000 bp作为启动子；用PLACE数据库对SiWRKY36启动子顺式作用元件进行分析；利用实时荧光定量PCR检测SiWRKY36在不同胁迫条件下（PEG、低温、NaCl、MeJA、ABA、GA和SA、H₂O₂）的表达模式；分别以8种胁迫处理的谷子cDNA作为模板，以谷子Si001873m.g为内参，以SYBR Green染料法进行real-time PCR。用实时荧光定量PCR仪进行PCR扩增；将SiWRKY36的cDNA序列连入带有CaMV 35S启动子的pBI121表达载体中，构建表达载体pBI121-SiWRKY36，转入农杆菌，侵染野生型拟南芥得到转基因株系。用T₃转SiWRKY36拟南芥植株进行抗性鉴定。【结果】谷子SiWRKY36全长1 485 bp，基因编码区包含UTR区和3个内含子以及4个外显子，与柳枝稷亲缘性最高，属于WRKY转录因子家族的第一类。SiWRKY36编码蛋白包含2个WRKY保守域，预测的SiWRKY36蛋白三级结构包含2个α螺旋结构和3个β折叠结构。启动子元件分析表明SiWRKY36包含ABA-responsive element（ABRE）、MYB、MYC、low-temperature-responsive element（LTRE）、GT-1等多种逆境胁迫应答元件。实时荧光定量PCR结果显示SiWRKY36对多种非生物胁迫和激素均有不同程度的响应，但在H₂O₂和低温处理下基因表达量无明显变化；亚细胞定位结果表明SiWRKY36蛋白主要定位于细胞核中。抗性鉴定结果显示，在不进行任何处理的MS培养基上，野生型拟南芥和过表达株系拟南芥的长势基本一致；在2% PEG的处理条件下，3个转基因株系的根长、根的总表面积和根的总体积要大于野生型拟南芥。【结论】SiWRKY36转基因植株可能对轻度干旱有一定的抗性。

关键词: 谷子, WRKY转录因子, 胁迫响应, 亚细胞定位, 抗逆性

Abstract: 【Objective】 Abiotic stresses seriously affect the plant growth development and the crop yield. WRKY transcription factors play a key role in the regulatory mechanism. This study provided experimental data for further study of the molecular characteristics and the function of SiWRKY36 gene. 【Method】 SiWRKY36 gene was isolated from the drought-treated foxtail millet transcriptome profile. Bioinformatics methods were used to analyze the molecular properties of the SiWRKY36 gene. Homologous sequences of SiWRKY36 were selected from the Phytozome program. Homologous analysis and multiple alignments were performed with MEGA 5. MEME and SMART online tools were used for protein sequence analysis. GSDS and PHYRE2 online tools were used to analyze gene structure and tertiary structure of SiWRKY36, respectively. Quantitative real-time PCR (qRT-PCR) was used to examine the expression pattern of SiWRKY36 gene in different abiotic stresses and phytohormone treatments. Full length SiWRKY36 cDNA was ligated into the vector to construct an expression vector for wild-type Arabidopsis plant. The pBI121-SiWRKY36 expression vector was introduced into Agrobacterium tumefaciens strain cells. Transgenic plants were used for resistance identification experiment. 【Result】 SiWRKY36 shared a high similarity with switchgrass and belonged to Group Ι of WRKY family including two WRKYGQK conserved domains. The predicted tertiary structure of SiWRKY36 contained two alpha helix and three beta folds. SiWRKY36 was mainly located in nucleus. The expression pattern showed that SiWRKY36 was involved in responses to various abiotic stresses and exogenous hormones, which was similar with the promoter analysis. The total root length, root surface area and root volume were slightly different between three SiWRKY36 lines and wild-type plants in drought treatment (2% PEG) in Arabidopsis plants. 【Conclusion】 SiWRKY36 transgenic plants have a certain resistance under mild drought conditions.

Key words: Foxtail millet, WRKY transcription factor, stress responses, subcellular localization, stress resistance

祖倩丽，尹丽娟，徐兆师，陈明，周永斌，李连城，马有志，闵东红，张小红. 谷子WRKY36转录因子的分子特性及功能鉴定[J]. 中国农业科学, 2015, 48(5): 851-860.

ZU Qian-li, YIN Li-juan, XU Zhao-shi, CHEN Ming, ZHOU Yong-bin, LI Lian-cheng, MA You-zhi, MIN Dong-hong, ZHANG Xiao-hong. Molecular Characteristics and Functional Identification of Foxtail Millet Transcription Factor WRKY36[J]. Scientia Agricultura Sinica, 2015, 48(5): 851-860.

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