小麦TaGRAS34-5A的克隆及耐热功能研究  

Cloning and Heat Tolerance Function of Wheat TaGRAS34-5A Gene

作　　者：刁邓超 李云丽 孟祥宇 季颂涵 孙玉晨 马学红 李杰 冯永佳 李春莲[1] 吴建辉 曾庆东[2,3] 韩德俊[1,3] 王长发[1,3] 郑炜君[1,3] DIAO DengChao;LI YunLi;MENG XiangYu;JI SongHan;SUN YuChen;MA XueHong;LI Jie;FENG YongJia;LI ChunLian;WU JianHui;ZENG QingDong;HAN DeJun;WANG ChangFa;ZHENG WeiJun(College of Agronomy,Northwest A&F University,Yangling 712100,Shaanxi;College of Plant Protection,Northwest A&F University,Yangling 712100,Shaanxi;Northwest A&F University/National Key Laboratory for Crop Stress Resistance and High-Efficiency Production,Yangling 712100,Shaanxi)

机构地区：[1]西北农林科技大学农学院,陕西杨凌712100 [2]西北农林科技大学植物保护学院,陕西杨凌712100 [3]西北农林科技大学/作物抗逆与高效生产全国重点实验室,陕西杨凌712100

出　　处：《中国农业科学》2025年第4期617-634,共18页Scientia Agricultura Sinica

基　　金：科技创新2030重大专项(2023ZD04026);陕西省重点研发计划一般项目(2023-YBNY-026)。

摘　　要：【目的】GRAS家族是植物特有的一类转录因子,在植物生长发育和响应逆境胁迫中发挥重要作用,明确GRAS家族基因在小麦耐热性中的作用,可为小麦耐热育种提供基因资源和理论支撑。【方法】基于TAM107和中国春苗期高温转录组筛选到一个潜在的热胁迫响应转录因子基因TaGRAS34-5A;对TaGRAS34-5A进行生物信息学分析并构建系统发育树,以明确其分子特征;采用实时荧光定量PCR(qRT-PCR)方法对TaGRAS34-5A在高温、脱落酸(ABA)、乙烯利(ETH)、水杨酸(SA)处理下的表达模式进行分析;利用小麦原生质体瞬时表达技术明确TaGRAS34-5A蛋白的亚细胞定位;利用酿酒酵母异源表达系统及BSMV:VIGS沉默技术对TaGRAS34-5A的耐热功能进行验证;利用酵母双杂交技术筛选TaGRAS34-5A潜在的互作蛋白,解析其耐热机理。【结果】TaGRAS34-5A含有一个典型的GRAS结构域,属于GRAS转录因子家族,定位于细胞核和细胞质。生物信息学分析表明,TaGRAS34-5A启动子中含有大量的激素响应元件和光响应元件,与TaSCL14、OsGRAS23、AtSCL14在亲缘关系上最近,暗示其潜在的应对氧化应激功能。在高温、ETH、ABA、SA处理下,TaGRAS34-5A均上调表达,分别在4、6、0.5和12 h后达到表达峰值,其中,受热胁迫和SA的诱导最强烈。酿酒酵母的耐热功能试验表明,TaGRAS34-5A的异源表达能够提高酿酒酵母的耐热性。BSMV:VIGS瞬时沉默试验结果显示,42℃高温处理后,TaGRAS34-5A沉默植株相较于对照,叶绿素含量下降、POD酶活降低、细胞过氧化程度增加、耐热性降低。初步耐热机理研究表明,TaGRAS34-5A具有强烈的转录自激活活性,TaGRAS34-5A可能通过与bZIP家族HBP-1b转录因子、E3泛素蛋白连接酶hel2等蛋白的相互作用,协同调控小麦的耐热能力。【结论】TaGRAS34-5A受热、ABA、ETH、SA诱导表达,编码的蛋白位于细胞核和胞质中,且具有转录激活活性,异源表达TaGRAS34-5A能够提高�【Objective】The GRAS family constitutes a unique class of plant-specific transcription factors that play a pivotal role in plant development and stress response.To elucidate the function of GRAS family genes in wheat heat tolerance,which can provide genetic resources and theoretical foundation for wheat heat-resistant breeding.【Method】A potential heat stress-responsive transcription factor gene,TaGRAS34-5A,was identified through transcriptome analysis of TAM107 and Chinese spring wheat seedlings under high-temperature conditions.Subsequently,a bioinformatics analysis was performed on TaGRAS34-5A,and a phylogenetic tree was constructed to elucidate its molecular characteristics.The expression pattern of TaGRAS34-5A under various stresses,including high temperature,abscisic acid(ABA),ethylene(ETH),and salicylic acid(SA)treatments,were examined using real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR)method.The subcellular localization of the TaGRAS34-5A protein was determined using wheat protoplast transient expression technique.Furthermore,the heat tolerance function of TaGRAS34-5A was validated using the heterologous expression system of Saccharomyces cerevisiae and the BSMV:VIGS(Barley stripe mosaic virus:Virus-Induced Gene Silencing)silencing technique.potential interacting proteins of TaGRAS34-5A were screened using yeast two-hybrid technology,and the heat tolerance function was verified,providing preliminary insights into its heat tolerance mechanism.【Result】TaGRAS34-5A,equipped with a characteristic GRAS domain and belongs to the GRAS transcription factor family,is localized to both the cell nucleus and cytoplasm.Bioinformatics analysis indicates that the TaGRAS34-5A promoter contains a large number of hormone response elements and light response elements,and it is most closely related to TaSCL14,OsGRAS23,and AtSCL14 in terms of phylogenetic relationships,suggesting its potential function in responding to oxidative stress.Its expression is upregulated under high-tempera

关 键 词：小麦 GRAS转录因子 热胁迫 基因沉默 蛋白互作 

分 类 号：S512.1[农业科学—作物学]