蒺藜苜蓿MtbHLH25基因克隆、亚细胞定位及表达分析

Cloning,subcellular localization,and expression analysis of MtbHLH25 gene in Medicago truncatula

[目的]bHLH转录因子数量众多,能够广泛参与植物的生长发育和逆境胁迫等过程。试验以蒺藜苜蓿R108为材料,克隆MtbHLH25基因并探究其功能,为研究蒺藜苜蓿bHLH转录因子的功能提供帮助。[方法]通过PCR扩增技术从蒺藜苜蓿中克隆MtbHLH25基因和启动子,构建酵母表达载体,并用LiAc转化法转移到Y2H Gold酵母菌株中进行酵母自激活检测,构建亚细胞定位载体,并通过冻融法转入农杆菌EHA105,菌液注射到烟草下表皮细胞后,用SP8激光共聚焦显微镜观察,通过实时荧光定量PCR技术研究MtbHLH25基因的时空表达水平。[结果](1)从蒺藜苜蓿中克隆出MtbHLH25基因和启动子,该基因总长882 bp,共编码293个氨基酸。启动子序列分析表明其包含ABA、MeJA、GA和SA等响应元件。(2)MtbHLH25蛋白与蚕豆和长柔毛野豌豆中bHLH蛋白高度同源。(3)MtbHLH25蛋白定位于细胞核。(4)酵母自激活检测结果显示MtbHLH25蛋白具有自激活活性。(5)MtbHLH25在蒺藜苜蓿根、茎、叶、花和果实中均有表达,其中在根中表达水平最高;外源SA、MeJA、ABA、GA以及盐胁迫使MtbHLH25基因表达量都呈下降趋势,表明SA、MeJA、ABA、GA以及盐胁迫对MtbHLH25基因表达起负调控作用。干旱胁迫能够显著诱导MtbHLH25基因表达量上调,说明该转录因子在干旱胁迫中起正调控作用。[结论]MtbHLH25基因对盐胁迫敏感,在干旱胁迫中发挥正调控作用;MtbHLH25蛋白具有自激活活性,对下游启动子调控的报告基因具有激活作用。

[Objective]bHLH transcription factors are numerous and widely participate in plant growth,development,and stress responses.The experiment used Medicago truncatula R108 as the material to clone the MtbHLH25 gene and preliminarily explored its function,which will help to further study the function of M.truncatula bHLH transcription factors.[Methods]MtbHLH25 gene and its promoter were cloned from M.truncatula using PCR technology.Yeast expression vector was constructed and transferred to Y2H Gold yeast strain using LiAc transformation method.Vectors for subcellular localization assays were constructed and transferred into Agrobacterium EHA105 through freeze-thaw method.The Agrobacterium was injected into tobacco epidermal cells and gene expression was observed using SP8 laser confocal microscopy.The spatiotemporal expression of MtbHLH25 in M.truncatula was observed using fluorescence quantitative PCR technology.[Results]The MtbHLH25 gene and its promoter were cloned from M.truncatula with 882 bp,encoding 293 amino acids.Promoter analysis revealed that it contains ABA response elements,MeJA response elements,GA response elements,and SA response elements.(2)The evolutionary tree showed that the MtbHLH25 protein was highly homologous with the bHLH proteins in Vicia faba and V.villosa Roth.(3)The MtbHLH25 protein was localized in the nucleus.(4)The yeast self-activation test showed that the MtbHLH25 protein has self-activation activity.(5)MtbHLH25 was expressed in roots,stems,leaves,flowers,and fruits of M.truncatula,with the highest expression in roots.Exogenous SA,MeJA,ABA,GA,and salt stress decreased the expression of MtbHLH25,indicating that SA,MeJA,ABA,GA,and salt stress had a negative regulatory effect on the expression of MtbHLH25.Drought stress increased the expression of MtbHLH25,indicating that this transcription factor might play a positive role in drought stress.[Conclusion]The MtbHLH25 gene may be sensitive to salt stress and play a positive regulatory role in drought stress.The MtbHLH25 protein has self-activating activity and may have an activating effect on the downstream reporter genes.