谷子转录因子基因SibZIP42在拟南芥中对高盐和ABA的响应

doi:10.3864/j.issn.0578-1752.2016.17.003

中国农业科学 ›› 2016, Vol. 49 ›› Issue (17): 3276-3286.doi: 10.3864/j.issn.0578-1752.2016.17.003

所属专题：谷子黍稷耐逆与遗传多样性

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

谷子转录因子基因SibZIP42在拟南芥中对高盐和ABA的响应

秦玉海^1,2，张小红³，冯露³，李微微²，徐兆师²，李连城²，周永斌¹，马有志²，刁现民²，贾冠清²，陈明²，闵东红¹

¹西北农林科技大学农学院/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作物遗传育种重点实验室，北京100081
³西北农林科技大学生命科学学院，陕西杨凌712100

收稿日期:2016-03-16 出版日期:2016-09-01 发布日期:2016-09-01
通讯作者: 闵东红，Tel：13609123593；E-mail：mdh2493@126.com。陈明，Tel：010-82108750；E-mail：chenming02@caas.cn
作者简介:秦玉海，E-mail：263820084@qq.com。张小红，E-mail：zhxh2493@126.com。秦玉海和张小红为同等贡献作者。
基金资助:
国家转基因生物新品种培育重大专项（2014ZX08002-003B）、陕西省科技统筹创新工程计划（2014KTZB02-01-01）、中国农业科学院创新工程

Response of Millet Transcription Factor Gene SibZIP42 to High Salt and ABA Treatment in Transgenic Arabidopsis

QIN Yu-hai^1,2, ZHANG Xiao-hong³, FENG Lu³, LI Wei-wei², XU Zhao-shi², LI Lian-cheng², ZHOU Yong-bin¹, MA You-zhi², DIAO Xian-min², JIA Guan-qing², CHEN Ming², MIN Dong-hong¹

¹ College of Agronomy, Northwest A＆F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi
²Institute of Crop Science, Chinese Academy of Agricultural Sciences/National Key Facility For Crop Gene Resource and Genetic Improvement/Key Laboratory of Biology and Genetic Improvement of Triticeae Crop, Ministry of Agriculture, Beijing 100081
³College of Life Science, Northwest A&F University, Yangling 712100, Shaanxi

Received:2016-03-16 Online:2016-09-01 Published:2016-09-01

摘要/Abstract

摘要： 【目的】分析谷子抗逆相关转录因子基因SibZIP42的特性和生物学功能，探讨SibZIP42提高植物耐盐性的调控途径，为作物抗逆分子育种提供新的候选基因。【方法】利用生物信息学方法分析谷子SibZIP42的特性：使用ClustalX 2.0和MEGA 5.05软件对谷子SibZIP42蛋白序列及其同源序列进行多序列比对，并构建系统进化树；从数据库Phytozome获取谷子SibZIP42上游2 000 bp作为启动子序列，在PLACE数据库对SibZIP42启动子顺式作用元件进行分析；使用NetPhos 2.0 Server数据库预测SibZIP42蛋白磷酸化位点；利用实时荧光定量PCR检测SibZIP42在不同胁迫条件下的表达模式；将SibZIP42与绿色荧光蛋白GFP融合表达，检测SibZIP42蛋白的亚细胞定位情况；构建植物表达载体pBI121-SibZIP42，转化拟南芥并检测转SibZIP42拟南芥的耐盐性及对ABA处理的敏感性。分析转SibZIP42拟南芥中ABA及脱水响应相关基因表达变化，分析SibZIP42调控植物耐盐性的作用机制。【结果】谷子SibZIP42全长546 bp，编码由181个氨基酸组成的亲水性蛋白，分子量约为20.3 kD，基因编码区包含1个外显子；系统进化树分析表明该基因位于bZIP基因家族的S亚组；SibZIP42与拟南芥AtbZIP42序列同源性最高；启动子元件分析表明，SibZIP42包含ABRE、MYB、MYC等多种逆境胁迫应答相关元件；磷酸化位点分析结果显示SibZIP42含有14个丝氨酸、4个酪氨酸和1个苏氨酸磷酸化位点；实时荧光定量PCR结果显示，SibZIP42对多种非生物胁迫均有不同程度的响应，在高盐、干旱（PEG）和ABA处理条件下表达量明显上升，SibZIP42在根部的表达量显著高于在茎及叶子中的表达；亚细胞定位结果表明，SibZIP42蛋白定位于细胞核中；基因功能分析结果显示，在正常MS培养基上，野生型拟南芥WT和SibZIP42转基因拟南芥的萌发率基本一致，在NaCl浓度为90、120和150 mmol·L^-1的MS培养基上，转基因拟南芥萌发率显著高于WT，在90 mmol·L^-1 NaCl处理条件下，转基因拟南芥的绿化率显著高于WT；在ABA浓度为0.5、1和2 μmol·L^-1的MS培养基上，转基因拟南芥的绿化率显著低于WT；下游基因检测结果表明，HIS1-3、RD29B和RAB18等ABA胁迫响应相关基因以及脱水响应相关基因AtPIP2A在转基因植株中表达量显著高于在WT中的表达，表明SibZIP42可能通过ABA信号途径提高植物对高盐胁迫的耐性。【结论】与WT相比，SibZIP42转基因拟南芥株系在种子萌发时期耐盐性显著提高。同时，在种子萌发后期SibZIP42转基因株系相比于WT对ABA处理的敏感性增强，SibZIP42可能通过ABA信号途径正向调控植物的耐盐性。

关键词: 谷子, bZIP类转录因子, 耐盐胁迫, ABA信号途径

Abstract: 【Objective】 This study was conducted to analyze the molecular characteristics and biological function of the transcription factor SibZIP42 in foxtail millet and to discuss the regulation pathway of SibZIP42 in improving salt tolerance. At the same time, the study would provide a potential gene for improving abiotic stress resistance in crop molecular breeding. 【Method】 Bioinformatics methods were used to analyze the molecular characteristics of SibZIP42. DNAMAN and MEGA 5.05 softwares were used to do multiple sequences alignment and construct the phylogenetic tree of SibZIP42. The 2 000 bp sequence upstream of SibZIP42 was downloaded as the promoter sequence from databases Phytozome, and the cis-acting elements of SibZIP42 promoter were analyzed in PLACE database. NetPhos 2.0 Server database was used to predict phosphorylation sites of SibZIP42 protein. The real-time PCR was used to analyze the expression patterns of SibZIP42 under various stress treatments. SibZIP42 was fused with GFP to detect its subcellular localization in protoplast cells. SibZIP42 was overexpressed in Arabidopsis to analyze its function under high salt and ABA stress conditions. 【Result】 The full length of SibZIP42 was 546 bp with one exons and encode a hydrophilic protein with 181 amino acid residues, and the protein molecular weight was about 20.3 kD. Phylogenetic tree showed that the gene is located in the S subgroup of bZIP family. It is the highest sequence homology with AtbZIP42. Promoter cis-acting element analysis showed that there are many stress-related response elements including ABRE, MYB and MYC in promoter sequence of SibZIP42. Phosphorylation site analysis showed that there are 14 serine, 4 tyrosine and 1 threonine phosphorylation sites in SibZIP42 protein sequence. The expression pattern analysis showed that SibZIP42 is involved in responses to various abiotic stresses and exogenous hormones. The gene expression profile results indicated that the expression level of SibZIP42 in root was higher than stem and leaf, and it was induced by drought, salt and ABA treatments in millet. The protein subcellular localization analysis revealed that SibZIP42 protein was localized in nucleus. Functional analysis of SibZIP42 showed that there is no significant difference of germination rate between transgenic lines and wild-type Arabidopsis WT on the normal MS medium, whereas on the MS culture medium with NaCl concentration of 90, 120 and 150 mmol·L^-1, germination rate of SibZIP42 transgenic Arabidopsis was significantly higher than WT. Under 90 mmol·L^-1 NaCl treatment condition, cotyledon green rate of SibZIP42 transgenic Arabidopsis was significantly higher than WT, whereas cotyledon green rate of SibZIP42 transgenic Arabidopsis was significantly lower than WT when the concentration of ABA was 0.5, 1 and 2 μmol·L^-1 in MS culture medium. Some ABA-related response genes including HIS1-3, RD29B and RAB18 and drought-related response gene including PIP2A were up-regulated in SibZIP42 transgenic Arabidopsis, which suggested that the overexpression of SibZIP42 improved salt stress tolerance in transgenic Arabidopsis by ABA signaling pathway. 【Conclusion】 The salt tolerance of SibZIP42 transgenic Arabidopsis was significantly stronger than WT during the period of seed germination, while SibZIP42 transgenic Arabidopsis seeds were more sensitive to ABA treatment than WT which indicated that SibZIP42 positively regulated salt tolerance in transgenic Arabidopsis by ABA signaling pathway.

Key words: foxtail millet (Setaria italica L.), bZIP transcription factor, salt stress tolerance, ABA signaling pathway

秦玉海，张小红，冯露，李微微，徐兆师，李连城，周永斌，马有志，刁现民，贾冠清，陈明，闵东红. 谷子转录因子基因SibZIP42在拟南芥中对高盐和ABA的响应[J]. 中国农业科学, 2016, 49(17): 3276-3286.

QIN Yu-hai, ZHANG Xiao-hong, FENG Lu, LI Wei-wei, XU Zhao-shi, LI Lian-cheng, ZHOU Yong-bin, MA You-zhi, DIAO Xian-min, JIA Guan-qing, CHEN Ming, MIN Dong-hong. Response of Millet Transcription Factor Gene SibZIP42 to High Salt and ABA Treatment in Transgenic Arabidopsis[J]. Scientia Agricultura Sinica, 2016, 49(17): 3276-3286.

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谷子转录因子基因SibZIP42在拟南芥中对高盐和ABA的响应

Response of Millet Transcription Factor Gene SibZIP42 to High Salt and ABA Treatment in Transgenic Arabidopsis

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