谷子WRKY36转录因子的分子特性及功能鉴定  

Molecular Characteristics and Functional Identification of Foxtail Millet Transcription Factor WRKY36

作　　者：祖倩丽 尹丽娟[2] 徐兆师[2] 陈明[2] 周永斌[1,2] 李连城[2] 马有志[2] 闵东红[1] 张小红[1] 

机构地区：[1]西北农林科技大学/旱区作物逆境生物学国家重点实验室,陕西杨凌712100 [2]中国农业科学院作物科学研究所/农作物基因资源与基因改良国家重大科学工程/农业部麦类生物学与遗传育种重点实验室,北京100081

出　　处：《中国农业科学》2015年第5期851-860,共10页Scientia Agricultura Sinica

基　　金：转基因生物新品种培育重大科技专项(2014ZX0800203B);陕西省科技计划项目(2013K02-01;2014K02-04-05);西北农林科技大学2014年唐仲英作物育种基金

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Si WRKY36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Si WRKY36;利用生物信息学的方法分析谷子Si WRKY36的分子特性;根据Si WRKY36蛋白序列进行同源性搜索,得到与谷子Si WRKY36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Si WRKY36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Si WRKY36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Si WRKY36上游2 000 bp作为启动子;用PLACE数据库对Si WRKY36启动子顺式作用元件进行分析;利用实时荧光定量PCR检测Si WRKY36在不同胁迫条件下(PEG、低温、Na Cl、Me JA、ABA、GA和SA、H2O2)的表达模式;分别以8种胁迫处理的谷子c DNA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Si WRKY36的c DNA序列连入带有Ca MV 35S启动子的p BI121表达载体中,构建表达载体p BI121-Si WRKY36,转入农杆菌,侵染野生型拟南芥得到转基因株系。用T3转Si WRKY36拟南芥植株进行抗性鉴定。【结果】谷子Si WRKY36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。Si WRKY36编码蛋白包含2个WRKY保守域,预测的Si WRKY36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Si WRKY36包含ABA-responsive element(ABRE)、MYB、MYC、low-temperature-responsive element(LTRE)�【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 Si WRKY36 gene. 【Method】 Si WRKY36 gene was isolated from the drought-treated foxtail millet transcriptome profile. Bioinformatics methods were used to analyze the molecular properties of the Si WRKY36 gene. Homologous sequences of Si WRKY36 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 Si WRKY36, respectively. Quantitative real-time PCR（q RT-PCR） was used to examine the expression pattern of Si WRKY36 gene in different abiotic stresses and phytohormone treatments. Full length Si WRKY36 c DNA was ligated into the vector to construct an expression vector for wild-type Arabidopsis plant. The p BI121-Si WRKY36 expression vector was introduced into Agrobacterium tumefaciens strain cells. Transgenic plants were used for resistance identification experiment. 【 Result 】 Si WRKY36 shared a high similarity with switchgrass and belonged to Group Ι of WRKY family including two WRKYGQK conserved domains. The predicted tertiary structure of Si WRKY36 contained two alpha helix and three beta folds. Si WRKY36 was mainly located in nucleus. The expression pattern showed that Si WRKY36 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 Si WRKY36 lines and wild-type plants in drought treatment（2% PEG） in Arabidopsis plants. 【Conclusion】 Si WRKY36 transgenic plants have a certain resistance under mild drought conditions.

关 键 词：谷子 WRKY转录因子 胁迫响应 亚细胞定位 抗逆性 

分 类 号：S515[农业科学—作物学]