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【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著高于WT,在90 mmol·L~(-1) Na Cl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2μmol·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At PIP2A在转基因植株中表达量显著高于在WT中的表达,表明Sib ZIP42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,Sib ZIP42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Sib ZIP42转基因株系相比于WT对ABA处理的敏感性增强,Sib ZIP42可能通过ABA信号途径正向调控植物的耐盐性。
【Objective】 The purpose of this study was to analyze the characteristics and biological functions of Sib ZIP42, a transcription factor involved in millet resistance, and to explore the regulation of Sib ZIP42 in improving plant salt tolerance and to provide novel candidate genes for crop resistance molecular breeding. 【Method】 The bioinformatics method was used to analyze the characteristics of Sib ZIP41. Multiple sequence alignment of the Sib ZIP41 protein sequence and its homologous sequences was carried out using Clustal X 2.0 and MEGA 5.05 software, and the phylogenetic tree was constructed. Phytozome The cis-acting element of the Sib ZIP42 promoter was analyzed in the PLACE database using a 2 000 bp upstream of the Milky Way Sib ZIP42 promoter as a promoter sequence; the prediction of the Sib ZIP42 protein phosphorylation site using the Net Phos 2.0 Server database; the detection of Sib ZIP42 by real-time fluorescence quantitative PCR Under different stress conditions; Sib ZIP42 and green fluorescent protein GFP fusion expression detected Sib ZIP42 protein subcellular localization; construct plant expression vector pB121-Sib ZIP42, transformed into Arabidopsis thaliana and detected Sib Sib41 Salt tolerance of Arabidopsis thaliana and its sensitivity to ABA treatment. Analysis of transcriptional changes of ABA and dehydration response genes in Arabidopsis thaliana transformed Sib ZIP42 and the mechanism by which Sib ZIP42 regulates salt tolerance in plants. 【Result】 The full length of Sib ZIP42 gene was 546 bp in length, encoding a 181 amino acid hydrophilic protein with a molecular weight of about 20.3 kD and a gene coding region containing one exon. Phylogenetic tree analysis showed that the gene was located in ZIP Sib ZIP42 had the highest homology with Atb ZIP42 in Arabidopsis thaliana. The analysis of promoter elements showed that Sib ZIP42 contained ABR, MYB, MYC and other elements related to stress response. The result of phosphorylation site analysis It shows that Sib ZIP42 contains 14 serine, 4 tyrosine and 1 threonine phosphorylation sites. Real-time PCR results showed that Sib ZIP42 responded differently to various abiotic stresses, The expression of Sib ZIP42 in roots was significantly higher than that in stems and leaves under drought (PEG) and ABA treatments. The subcellular localization of Sib ZIP42 protein was located in the nucleus. The results of gene function analysis The results showed that the germination rate of wild-type Arabidopsis thaliana WT and Sib ZIP42 transgenic Arabidopsis thaliana was the same on the normal MS medium. MS medium with NaCl concentrations of 90, 120 and 150 mmol·L -1 On the transgenic Arabidopsis germination rate significantly In WT, the greening rate of transgenic Arabidopsis thaliana was significantly higher than that of WT under 90 mmol·L -1 NaCl treatment. In the MS culture with ABA concentrations of 0.5, 1 and 2 μmol·L -1, , The greening rate of transgenic Arabidopsis thaliana was significantly lower than that of WT. The downstream gene test showed that the ABA stress response related genes such as HIS1-3, RD29B and RAB18 and the expression level of At PIP2A in transgenic plants were significantly higher than that of transgenic plants Expression in WT showed that Sib ZIP42 may enhance plant tolerance to high salt stress through the ABA signaling pathway. 【Conclusion】 Compared with WT, the salt tolerance of Sib ZIP42 transgenic Arabidopsis plants was significantly increased during the seed germination period. At the same time, Sib ZIP42 transgenic lines were more sensitive to ABA than WT at the late germination stage, and Sib ZIP42 could positively regulate the salt tolerance of plants through the ABA signaling pathway.