日本结缕草ZjERF1的克隆、转录激活活性、亚细胞定位及表达分析  

Molecular cloning, transcriptional activation, subcellular localization analysis and expression characterization of ZjERF1 from Zoysia japonica

作　　者：滕珂 张蕊 檀鹏辉 岳跃森 范希峰[1] 武菊英[1] TENG Ke;ZHANG Rui;TAN Peng-hui;YUE Yue-sen;FAN Xi-feng;WU Ju-ying(Beijing Research and Development Center for Grass and Environment, Beijing 100097, China;Institute of Turfgrass Science, Beijing Forestry University, Beijing 100083, China)

机构地区：[1]北京草业与环境研究发展中心,北京100097 [2]北京林业大学草坪研究所,北京100083

出　　处：《草业学报》2019年第6期56-65,共10页Acta Prataculturae Sinica

基　　金：北京市农林科学院科技创新能力建设专项(KJCX20161502-1、KJCX20170110);中国博士后基金(2017M620677);北京市博士后基金(2017-ZZ-087);北京市科技计划(D171100007217001)资助

摘　　要：ERF转录因子是植物体内最大的一类转录因子之一,在调控植物生长发育和响应环境胁迫等方面发挥着重要的作用。研究利用RACE技术,从日本结缕草中克隆到ZjERF1基因(GenBank登录号为MH294481),开放阅读框为630 bp,编码209个氨基酸残基,含有1个高度保守的AP2结构域,属于典型的ERF转录因子。利用染色体步移技术,成功获得ATG上游1581 bp的启动子序列,生物信息学分析表明其中含有多个响应MeJA等激素和非生物胁迫的作用元件。为分析其转录激活特性,构建了pGBKT7-ZjERF1载体,转化Y2HGold酵母感受态细胞,结果表明其具有较强的转录激活活性。为探析其亚细胞定位情况,成功构建35S::ZjERF1:YFP载体,本生烟草瞬时表达结果证明ZjERF1定位于细胞核。为进一步研究ZjERF1的表达特征,利用实时荧光定量的方法进行了验证,结果表明:ZjERF1在日本结缕草茎中表达量最高,并且与叶片的衰老程度呈正相关性;此外ZjERF1的表达可受200μmol·L^-1 ET、10μmol·L^-1 MeJA和300 mmol·L^-1 NaCl处理的诱导,但受到20%PEG4000的抑制。研究表明ZjERF1是一个可参与多种信号转导途径的优良转录因子,为进一步探索ZjERF1基因的功能及其调控机制奠定了基础。The ERF transcription factors (TFs) are one of the largest groups in plants and play important roles in plant developmental progress and responses to abiotic stresses. In this study, one novel ERF gene, ZjERF1 (GenBank No. MH294481), was isolated from Zoysia japonica using the RACE method. The open reading frame of ZjERF1 is 630 bp in length, encoding 209 amino acids. One highly conserved AP2 domain was found in ZjERF1, indicating ZjERF1 was a typical ERF TF. By genome walking, a 1581 bp upstream sequence from the ATG of ZjERF1 was obtained. Bioinformatics analysis revealed that there were several cis-elements in response to MeJA and abiotic stresses in the promoter. To investigate the transcriptional activation character, a pGBKT7-ZjERF1 vector was constructed and then was transformed into Y2HGold yeast cells;the results showed that ZjERF1 had strong transcriptional activity. To reveal the subcellular localization character, 35S::ZjERF1:YFP was generated. The transient expression analysis in Nicotiana benthamiana demonstrated that ZjERF1 was localized in the nucleus. To further explore the expression characteristics, real-time quantitative PCR was carried out. The results showed that ZjERF1 was expressed most abundantly in the stem and its transcriptional abundance was positively correlated with leaf senescence. Moreover, its expression could be induced by 200 μmol·L^-1 ET, 10 μmol·L^-1 MeJA or 300 mmol·L^-1 NaCl but was suppressed by 20% PEG4000. Taken together, these findings proves that ZjERF1 is a functional TF involved in various signaling pathways and paves the way for further study of the function of ZjERF1 and its regulatory mechanism in Z. japonica.

关 键 词：日本结缕草 ERF转录因子 转录激活 亚细胞定位 表达分析 

分 类 号：S688.4[农业科学—观赏园艺]