青杄转录因子PwERF8及其启动子序列的克隆与分析  

Cloning and Analysis of a Transcription Factor PwERF8 and the Promoter Sequences in Picea wilsonii

作　　者：张鹤华 刘嘉欣 罗朝兵[1] 张凌云[1] Zhang Hehua;Liu Jiaxin;Luo Chaobing;Zhang Lingyun(Key Laboratory of Forestry Silviculture and Conservation of Ministry of Education Beijing Forestry University Beijing 100083)

机构地区：[1]北京林业大学,森林培育与保护教育部重点实验室,北京100083

出　　处：《林业科学》2018年第3期48-60,共13页Scientia Silvae Sinicae

基　　金：转基因生物新品种培育重大专项“抗逆和抗除草剂关键基因克隆及功能验证”(2016ZX08009003-002)。

摘　　要：【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。从青杄中克隆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,为深入了解青杄的耐逆调控机制提供理论基础。【方法】通过RACE-PCR技术获得青杄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1 191 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分别处理青杄幼苗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�【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 PwERF 8 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 PwERF 8 was cloned from P.wilsonii by RACE-PCR technique.The promoter sequence of PwERF 8 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-PwERF 8 promoter∷GUS was transferred into tobacco leaves by agrobacterium-mediated method.The PGBKT7-PwERF 8 was transformed into AH109 yeast strain to verify its transcriptional activation activity.To investigate the subcellular location of PwERF8,the construct encoding 35S∷PwERF 8-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 PwERF 8 under abiotic stress.【Result】The full-length coding of PwERF 8 was 1 190 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 PwERF 8 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 PwERF 8 contained cis-acting elements such as GA,ABA,JA and SA.Furthermore,GUS

关 键 词：青杄 ERF转录因子 激素 非生物逆境胁迫 启动子 基因表达 

分 类 号：S718.46[农业科学—林学]