小麦热胁迫响应基因TaMYB165的克隆与功能分析

Cloning and functional analysis of heat-responsive gene TaMYB165 in wheat

【目的】MYB转录因子在植物生长发育和抗逆过程中起着重要作用,克隆小麦MYB基因并对其功能进行研究,对植物MYB转录因子的调控机制具有重要意义。【方法】利用Affymetrix小麦芯片系统,分析2个小麦品种中国春(热敏感)和TAM107(耐热)在不同热胁迫处理下的转录谱,从热胁迫上调表达的EST序列中克隆得到小麦耐热相关TaMYB165基因,并对该基因的表达特性及功能进行研究;利用实时定量RT-PCR技术分析小麦不同发育时期、不同组织器官热胁迫处理下TaMYB165基因的动态表达模式,构建超表达载体并转化拟南芥,对获得的转基因拟南芥株系进行耐热性鉴定。【结果】克隆获得1个小麦MYB转录因子家族基因TaMYB165,该基因ORF长度为847 bp,编码282个氨基酸。同源序列分析表明,TaMYB165与高粱SORBIDRAFT 03g040120亲缘关系最近(73.2%),与水稻MYB蛋白和拟南芥AtMYB165的相似性分别为70.37%和33.0%。qRT-PCR结果显示,TaMYB165在小麦苗期和灌浆期都受热胁迫诱导表达,只是响应的早晚有所不同。在拟南芥中过量表达TaMYB165,转基因株系热胁迫后成活率提高,细胞膜热稳定性增强。【结论】TaMYB165是小麦热胁迫响应的重要转录因子,可作为小麦耐热育种的重要候选基因。

【Objective】 MYB transcription factors play diverse roles in plant growth,development and stress responses.Cloning and functional research of MYB gene in wheat are of great significance to understand the regulation mechanism of MYB transcription factors.【Method】 In this study, we analyzed the transcriptional profiles of heat resistance of two wheat genotypes of Chinese spring(heat susceptible) and TAM107(heat tolerant) under different heat stress treatments using Affymetrix wheat chip system.A heat induced gene TaMYB165 was cloned based on the up-regulated EST,and the expression characterization and function of the gene were studied.The dynamic expression pattern of TaMYB165 in different tissues of wheat at different development stages was analyzed by qRT-PCR.Transgenic lines were obtained by constructing overexpression vectors and transforming Arabidopsis thaliana,and heat resistance of transgenic lines were identified.【Result】 A heat induced transcription factor gene TaMYB165 was cloned using the approach of silicon cloning with ORF length of 847 bp,encoding a protein of 282 amino acids.Homologous sequence analysis indicated that TaMYB165 had the highest similarity with SORBIDRAFT03 g040120(73.2%) from Sorghum bicolor,and the similarity with MYB proteins from rice and Arabidopsis AtMYB165 were as high as 70.37% and 33.0%,respectively.Real-time quantitative RT-PCR results revealed that the genes were induced by heat stress in wheat seedling and filling stages.TaMYB165 were overexpressed in Arabidopsis thaliana,and the results demonstrated that the survival rate of transgenic lines was improved and the thermal stability of cell membrane was significantly enhanced.【Conclusion】 TaMYB165 is an important transcription factor,and it can be used as a promising candidate gene for heat resistance breeding of wheat.