小麦<i>TaWRKY70</i>的克隆、亚细胞定位与表达分析

doi:10.7668/hbnxb.20195906

摘要/Abstract

摘要：

为系统解析WRKY转录因子家族成员在小麦生长发育调控及非生物胁迫应答中的作用,研究了TaWRKY基因在干旱、高盐、低温等逆境条件下的表达模式特征。以普通小麦品种中国春为材料,通过分子克隆技术获得TaWRKY70基因,其编码区全长885 bp,编码294个氨基酸的不稳定亲水性蛋白。生物信息学分析表明,该蛋白具有典型的WRKYGQK保守结构域及C₂HC型锌指结构,符合第Ⅲ类WRKY转录因子的分类特征。通过启动子顺式作用元件分析发现,TaWRKY70启动子区域存在参与茉莉酸甲酯响应、脱落酸响应、乙烯响应等调控元件。基因共表达分析表明,TaWRKY70与小麦对激素响应、对微生物的防御反应、对寒冷等非生物胁迫的应答等多个抗逆过程相关。系统进化树分析表明,TaWRKY70蛋白与大麦、玉米、高粱和谷子等禾本科作物的WRKY70亲缘关系较近。亚细胞定位结果证明,TaWRKY70定位于细胞核,符合转录因子特征。通过表达模式分析发现,TaWRKY70在小麦根、茎、叶、幼穗和籽粒中均有表达,并且在根和叶中表达较高。在非生物胁迫下,TaWRKY70在脱落酸和低温处理下表达下调,在水杨酸、NaCl、PEG6000和高温处理下表达上调。综上,通过克隆小麦TaWRKY70基因并分析其表达模式,为下一步解析TaWRKY70参与小麦抗逆的分子机制提供了基础。

关键词: 小麦, TaWRKY70, 基因克隆, 亚细胞定位, 表达分析

Abstract:

To elucidate the role of WRKY transcription factor family members in the dynamic regulation of wheat growth and development,as well as in responses to abiotic stresses,this study investigated the expression patterns of TaWRKY gene under drought,high salinity,and low-temperature stress conditions.Using the common wheat cultivar Chinese Spring as the experimental material,we obtained the TaWRKY70 gene through molecular cloning.The coding sequence of TaWRKY70 was 885 bp in length,encoding a 294-amino-acid hydrophilic and unstable protein.Bioinformatics analysis revealed that the protein possessed a typical WRKYGQK conserved domain and a C₂HC-type zinc finger structure,classifying it as a Group Ⅲ WRKY transcription factor.Cis-regulatory element analysis of the TaWRKY70 promoter region identified regulatory elements involved in responses to methyl jasmonate,abscisic acid,and ethylene.Co-expression gene analysis suggested that TaWRKY70 was associated with multiple stress response processes in wheat,including hormone signaling,defense against microbial pathogens,and responses to cold stress.Phylogenetic analysis indicated that TaWRKY70 shared a close evolutionary relationship with WRKY70 proteins from other Poaceae species,such as barley,maize,sorghum,and foxtail millet.Subcellular localization experiment further confirmed that TaWRKY70 was localized in the nucleus,consistent with the characteristics of a transcription factor.Expression pattern analysis showed that TaWRKY70 was expressed in wheat roots,stems,leaves,young spikes,and grains,with higher expression levels observed in roots and leaves.Under abiotic stress conditions,TaWRKY70 expression was downregulated in response to abscisic acid and low-temperature treatments but upregulated under salicylic acid,NaCl,PEG6000,and high-temperature treatments.In conclusion,the cloning of TaWRKY70 gene and analysis of its expression pattern provide a basis for the next step to analyze the molecular mechanism of TaWRKY70 involved in wheat stress resistance.

Key words: Wheat, TaWRKY70, Gene cloning, Subcellular localization, Expression analysis

中图分类号:

卢振华, 梁晨, 张丽, 佟可心, 陈小强, 李明, 丁博, 邱丽娜, 谢晓东, 王俊斌. 小麦TaWRKY70的克隆、亚细胞定位与表达分析[J]. 华北农学报, 2025, 40(5): 12-19. doi: 10.7668/hbnxb.20195906.

LU Zhenhua, LIANG Chen, ZHANG Li, TONG Kexin, CHEN Xiaoqiang, LI Ming, DING Bo, QIU Lina, XIE Xiaodong, WANG Junbin. Cloning,Subcellular Localization and Expression Analysis of TaWRKY70 in Wheat[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(5): 12-19. doi: 10.7668/hbnxb.20195906.

http://www.hbnxb.net/CN/Y2025/V40/I5/12

图/表 9

图1 小麦TaWRKY70基因PCR扩增结果 M.2K PlusⅡ DNA Marker;1,2,3.TaWRKY70的片段;4.阴性对照(H₂O)。

Fig.1 PCR amplification of TaWRKY70 in wheat M.2K PlusⅡ DNA Marker;1,2,3.Fragments of TaWRKY70; 4.Negative control(H₂O).

图2 TaWRKY70编码的氨基酸序列及保守结构域分析阴影部分为WRKY保守结构域。其中,横线部分为WRKYGQK核心序列,方框指示C₂HC型锌指结构。

Fig.2 Analysis of amino acid sequences and conserved domains encoded by TaWRKY70 The shaded area represents the conserved WRKY domain.The sequence with underline means WRKYGQK core sequence and the C₂HC zinc-finger structure are boxed.

表1 TaWRKY70基因启动子顺式作用元件及功能注释

Tab.1 Cis-acting elements and functional annotations of TaWRKY70 promoter

顺式作用元件 Cis-acting elements	数量 Number	功能 Function
TGA-element	1	生长素响应元件
TGACG-motif	8	茉莉酸甲酯响应元件
CGTCA-motif	8	茉莉酸甲酯响应元件
TCA-element	1	水杨酸响应元件
CCGTCC-box	8	乙烯响应元件
TATC-box	1	赤霉素响应元件
ABRE	1	ABA响应元件
TCCC-motif	3	光响应元件
Sp1	2	光响应元件
Box 4	2	光响应元件
MBS	1	与干旱诱导相关的MYB结合位点
GCN4_motif	1	参与胚乳表达的顺式调控元件
GC-motif	3	参与缺氧特异性诱导的增强子样元件

图3 TaWRKY70共表达基因的GO富集分析颜色表示富集程度;大小表示富集的基因数量。

Fig.3 GO enrichment analysis of TaWRKY70 co-expression genes Color represents the enrichment level;size represents the number of enriched genes.

图4 TaWRKY70与其他植物同源WRKY70的系统进化树分析

Fig.4 Phylogenetic tree of TaWRKY70 and its homologous WRKY70 proteins

图5 TaWRKY70亚细胞定位

Fig.5 Subcellular localization of TaWRKY70

图6 TaWRKY70基因组织特异性表达分析不同小写字母表示差异显著(P<0.05)。图8同。

Fig.6 Tissue-specific expression analysis of the TaWRKY70 gene Different lowercase letters indicate significant differences(P<0.05).The same as Fig.8.

图7 TaWRKY70基因在不同非生物胁迫下的表达热图

Fig.7 Relative expression heatmap of TaWRKY70 gene under different abiotic stresses

图8 不同非生物胁迫处理条件下TaWRKY70表达模式分析

Fig.8 Analysis of expression patterns of TaWRKY70 under different stress treatment conditions

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小麦TaWRKY70的克隆、亚细胞定位与表达分析

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