林业科学2018，Vol.54Issue(3)：48-60,13.DOI:10.11707/j.1001-7488.20180306
青杆转录因子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
摘要
Abstract
[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.关键词
青杆/ERF转录因子/激素/非生物逆境胁迫/启动子/基因表达Key words
Picea wilsonii/ERF transcription factor/hormone/abiotic stress/promotor/gene expression分类
农业科技引用本文复制引用
张鹤华,刘嘉欣,罗朝兵,张凌云..青杆转录因子PwERF8及其启动子序列的克隆与分析[J].林业科学,2018,54(3):48-60,13.基金项目
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). （2016ZX08009003-002）
