[目的]AP2/ERF是植物的转录因子家族之一,广泛参与生物胁迫和非生物胁迫过程.从青杆中克隆ERF类转录因子PwERF8及其启动子序列,研究PwERF8基因的表达特性、启动子序列功能,并进一步分析其对非生物胁迫的响应模式,为深入了解青杆的耐逆调控机制提供理论基础.[方法]通过RACE-PCR技术获得青杆PwERF8编码区全长序列.通过染色体步移技术克隆PwERF8启动子序列.通过PlantCARE在线软件和BDGP在线软件预测启动子序列上的顺式作用元件、基础启动子和转录起始位点,构建pBI121-PwERF8 promoter∷GUS启动子表达载体,采用农杆菌注射法瞬时转化烟草叶片来验证启动子的功能.构建PGBKT7-PwERF8载体,转化AH109酵母菌株验证其转录激活活性.构建35S∷PwERF8-GFP融合表达载体,通过PEG介导瞬时转化拟南芥原生质体进行基因表达的亚细胞定位分析,应用RT-qPCR技术分析该基因的组织特异性和在非生物胁迫下的时空表达特征.[结果]PwERF8基因的编码区全长序列共1191 bp,开放阅读框共765 bp,开放阅读框翻译成255个氨基酸.在肽链的N端具有1个保守的由58个氨基酸组成的AP2结构域,在C端含有1个EAR转录抑制基序(DLNLPP).组织特异性表达分析表明,PwERF8在茎、根、针叶、花粉、种子中均有表达,但在花粉中,PwERF8的表达量最高,其次为种子,在茎中的表达量最少.酵母单杂交试验表明,PwERF8蛋白不具有转录激活活性.亚细胞定位分析发现PwERF8蛋白主要定位于细胞核.将启动子序列进行在线分析显示PwERF8含有GA、ABA、JA和SA等激素的顺式作用元件.进一步在烟草中瞬时过表达PwERF8启动子并用GUS染色显示,PwERF8启动子能响应GA、ABA、MeJA和SA等外源激素的处理.GA、ABA、MeJA和SA分别处理青杆幼苗3、6、12 h后,PwERF8的表达量均显著高于对照,干旱、4℃和42℃处理均能诱导PwERF8表达,但盐胁迫不能诱导其表达.[结论]青杆转录因子PwERF8参与了GA、ABA、JA和SA激素的信号通路,广泛响应干旱、温度逆境等非生物胁迫,且可能作为一个转录抑制子在细胞核中发挥功能.%[Objective] AP2/ERF (APETALA2/ethylene-responsive factor) is one of the largest transcription factors in plants and widely involved in biotic stresses and abiotic stresses.The aims were to study the expression characteristics and promoter sequence function of PwERF8 in Picea wilsonii and clarify the expression pattern in response to abiotic stress.The study would provide a basis for further understanding the regulation mechanism of stress resistance.[Method] The full coding sequence of PwERF8 was cloned from P.wilsonii by RACE-PCR technique.The promoter sequence of PwERF8 was cloned by genome walking.The cis-acting elements,BCP region and transcriptional start site were predicted by PlantCARE and BDGP online software.To characterize the function of promotor,the pBI121-PwERF8 promoter ∷ GUS was transferred into tobacco leaves by agrobacterium-mediated method.The PGBKT7-PwERF8 was transformed into AH109 yeast strain to verify its transcriptional activation activity.To investigate the subcellular location of PwERF8,the construct encoding 35S ∷ PwERF8-GFP fusion protein was transferred into Arabidopsis thaliana protoplast by PEG-mediated method.RT-qPCR was performed to analyze the tissue expression specificity and the dynamic expression of PwERF8 under abiotic stress.[Result] The full-length coding of PwERF8 was 1190 bp containing a 765 bp open reading frame flanked and encoded 255 amino acids.It contains one AP2 domain consisting of 58 amino acids in the N-terminal and one EAR motif (DLNLPP) in the C-terminal.The tissue-specific expression analysis showed that PwERF8 were all expressed in stem,root,needle,pollen and seed,however,the expression level in pollen was the highest,followed by seed.Single cross of yeast showed no transcriptional activation activity of PwERF8 protein.The subcellular localization analysis showed that PwERF8 was mainly localized in the nucleus.The online analysis showed that the promoter sequence of PwERF8 contained cis-acting elements such as GA,ABA,JA and SA.Furthermore,GUS color reaction experiment showed that PwERF8 promoter sequences with cis elements could respond to GA,ABA,JA and SA hormones.The expression of PwERF8 under GA,ABA,MeJA and SA hormone treatments after 3 h,6 h and 12 h were significantly higher than those in the control.The expression of PwERF8 under stress treatments showed that the expression of PwERF8 was induced by drought,4℃ and 42℃,respectively,but not by the salt stress.[Conclusion] Picea wilsonii transcription factor PwERF8 is involved in the signaling pathways of GA,ABA,JA and SA hormones and extensively responds to abiotic stresses such as drought,temperature stress.It probably plays a major role in the nucleus as a transcriptional repressor.
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