牡丹PsZFP1转录因子的特性及表达分析

Characteristic and Expression Analysis of a Transcription Factor PsZFP1 in Tree Peony

含保守锌指结构域的锌指蛋白(ZFP)是一类在调控植物生长发育和逆境响应过程中具有重要作用的转录因子,为了探究转录因子PsZFP1在牡丹休眠解除过程中的功能和作用机制,以牡丹鲁菏红花芽的RNA为模板,通过5′-RACE扩增PsZFP1的全长cDNA序列,利用EditSeq和MEGA 6.0进行氨基酸序列和系统进化树分析,通过烟草叶片瞬时转化分析PsZFP1的亚细胞定位,构建pGBKT7-PsZFP1重组质粒分析PsZFP1蛋白的转录激活活性,并通过酵母单杂鉴定PsZFP1与PsMPT的启动子是否结合;利用qRT-PCR分析PsZFP1基因表达特性,构建重组质粒pET28a+-PsZFP1转化大肠杆菌BL21进行原核表达。结果显示:牡丹PsZFP1基因的全长cDNA序列为1313 bp,其最大开放阅读框为915 bp,编码304个氨基酸;PsZFP1蛋白为亲水性的稳定蛋白,具有CCCH保守结构域,与葡萄VvZFP的亲缘关系最近;PsZFP1蛋白位于细胞核中,具有转录激活活性,具备转录因子特性;PsZFP1可以结合到PsMPT启动子的MYC元件上;qRT-PCR结果显示,低温21 d,PsZFP1的表达量最高,28 d显著下降;重组质粒pET28a+-PsZFP1在BL21中表达,经Ni-NTA柱纯化获得纯度较好的可溶性PsZFP1蛋白。该研究证明PsZFP1为含锌指结构的转录因子,受低温累积诱导,进而上调PsMPT基因表达,促进牡丹花芽能量代谢。

Zinc finger proteins(ZFPs),which containing conserved zinc finger domains,play important roles in the plant growth and development as well as in stress responses.In order to further explore the function and mechanism of PsZFP1 in the process of breaking dormancy in tree peony,the full-length cDNA sequence of PsZFP1 was cloned from Paeonia suffruticosa Luhehong by the 5′-RACE.After that,the amino acid sequence and phylogenetic tree of PsZFP1 were analyzed by the EditSeq and MEGA 6.0 software.Then,Agrobacterium tumefaciens-mediated transient transformation of tobacco was used to investigate subcellular localization of PsZFP1,and the pGBKT7-PsZFP1 recombinant plasmid was constructed for the transcriptional activity analysis.Yeast one hybridization was used to analysis the interaction between PsZFP1 and PsMPT promoter.In addition,the qRT-PCR was performed for the expression analysis of PsZFP1 in the flower buds of tree peony along with chilling duration process.The pET28a+-PsZFP1 recombinant plasmid was also constructed and expressed in BL21 in order to obtain PsZFP1 protein.The results showed that the PsZFP1 cDNA was 1313 bp,and its open reading maximum frame was 915 bp,which encoded a protein with 304 amino acid.The PsZFP1 protein was a hydrophilic and stable protein,which contained a conserved CCCH domain,and shared a high degree of sequence similarity with a VvZFP from grape.The PsZFP1 protein was located in the nucleus,and was a transcriptional factor which had transcriptional activation activity.Further yeast one hybridization analysis confirmed that PsZFP1 could bind to the MYC element of PsMPT promoter.In addition,the expression of PsZFP1 gene increased in the flower buds of tree peony along with chilling duration process,and the highest expression of PsZFP1 gene was presented at 21 d chilling and decreased significantly at 28 d chilling.Moreover,the PsZFP1 protein was expressed in prokaryotic cells,and the purified protein PsZFP1 was then obtained by Ni-NTA affinity chromatography and detected by SDS-PAGE.Taken together,we identified a zinc finger transcriptional factor,which could be induced by chilling accumulation and regulate PsMPT expression for energy metabolism during chilling induced dormancy release.The results were benefit to further analyze the function of PsZFP1 and its regulation to endodormancy.