摘要
【目的】分析谷子抗逆相关转录因子基因Sib ZIP42的特性和生物学功能,探讨Sib ZIP42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Sib ZIP42的特性:使用Clustal X 2.0和MEGA 5.05软件对谷子Sib ZIP42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Sib ZIP42上游2 000 bp作为启动子序列,在PLACE数据库对Sib ZIP42启动子顺式作用元件进行分析;使用Net Phos 2.0 Server数据库预测Sib ZIP42蛋白磷酸化位点;利用实时荧光定量PCR检测Sib ZIP42在不同胁迫条件下的表达模式;将Sib ZIP42与绿色荧光蛋白GFP融合表达,检测Sib ZIP42蛋白的亚细胞定位情况;构建植物表达载体p BI121-Sib ZIP42,转化拟南芥并检测转Sib ZIP42拟南芥的耐盐性及对ABA处理的敏感性。分析转Sib ZIP42拟南芥中ABA及脱水响应相关基因表达变化,分析Sib ZIP42调控植物耐盐性的作用机制。【结果】谷子Sib ZIP42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20.3k D,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b ZIP基因家族的S亚组;Sib ZIP42与拟南芥Atb ZIP42序列同源性最高;启动子元件分析表明,Sib ZIP42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Sib ZIP42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,Sib ZIP42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,Sib ZIP42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,Sib ZIP42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Sib ZIP42转基因拟南芥的萌发率基本一致,在Na Cl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�
【Objective】 This study was conducted to analyze the molecular characteristics and biological function of the transcription factor Sib ZIP42 in foxtail millet and to discuss the regulation pathway of Sib ZIP42 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 Sib ZIP42. DNAMAN and MEGA 5.05 softwares were used to do multiple sequences alignment and construct the phylogenetic tree of Sib ZIP42. The 2 000 bp sequence upstream of Sib ZIP42 was downloaded as the promoter sequence from databases Phytozome, and the cis-acting elements of Sib ZIP42 promoter were analyzed in PLACE database. Net Phos 2.0 Server database was used to predict phosphorylation sites of Sib ZIP42 protein. The real-time PCR was used to analyze the expression patterns of Sib ZIP42 under various stress treatments. Sib ZIP42 was fused with GFP to detect its subcellular localization in protoplast cells. Sib ZIP42 was overexpressed in Arabidopsis to analyze its function under high salt and ABA stress conditions. 【Result】 The full length of Sib ZIP42 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 k D. Phylogenetic tree showed that the gene is located in the S subgroup of b ZIP family. It is the highest sequence homology with Atb ZIP42. Promoter cis-acting element analysis showed that there are many stress-related response elements including ABRE, MYB and MYC in promoter sequence of Sib ZIP42. Phosphorylation site analysis showed that there are 14 serine, 4 tyrosine and 1 threonine phosphorylation sites in Sib ZIP42 protein sequence. The expression pattern analysis showed that Sib ZIP42 is involved in responses to various abiotic stresses and exogenous hormones. The gene expression profile results indicated that the expression level of Sib ZIP42 in root was higher t
出处
《中国农业科学》
CAS
CSCD
北大核心
2016年第17期3276-3286,3473-3475,共11页
Scientia Agricultura Sinica
基金
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
关键词
谷子
bZIP类转录因子
耐盐胁迫
ABA信号途径
foxtail millet（Setaria italica L.）
b ZIP transcription factor
salt stress tolerance
ABA signaling pathway
