| Maize（Zea mays L.）is the largest grain crop in China and an important feed and industrial raw material.The healthy and sustainable development of the corn industry is of great significance for ensuring national food security.During the process of plant growth and development,plants will encounter various stress conditions,such as drought,waterlogging,high temperature,saline-alkali,and low temperature,which can seriously affect their normal growth.To cope with complex and ever-changing adverse environ-ments,plants will establish many response mechanisms to regulate their physiological activities.Transcription factors（TF）play an important regulatory role in plant growth,development,and stress response.AP2/ERF family transcription factors are a kind of transcription factor superfamily widely existing in plants,which are widely involved in plant growth and development regulation（including plant height,root development,flower development,seed germination,organ size）,resistance to biotic and abiotic stresses,and plant hormone signal transduction pathways.Therefore,the exploration and functional research of maize AP2/ERF transcription factor genes have important guiding significance for cultivating new maize varieties with high and stable yield,stress re-sistance,and wide adaptability through modern molecular biology techniques.In the previous research work,we performed root phenotypic identification and tran-scriptome sequencing（RNA-seq）analysis on four critical periods（six-leaf stage（V6）,twelve-leaf stage（V12）,tasseling stage（VT）and milk ripening stage（R3））during maize growth and development.We screened 26 transcription factor genes that were signifi-cantly differentially expressed during adjacent periods,including an AP2/ERF family member gene ZmEREB211.In this study,ZmEREB211 gene was cloned.Through real-time quantitative PCR,subcellular localization,phenotypic identification of transgenic Arabidopsis lines,yeast two-hybrid and other experiments,the bioinformatics character-istics,tissue differential expression patterns,subcellular localization and response to stress of ZmEREB211 gene were preliminarily studied and analyzed.It provides new ideas and new gene resources for further analysis of its biological function in maize,as well as new germplasm creation and genetic improvement of maize.The main experimental re-sults are as follows:（1）Using maize inbred line B73 as the material,the ZmEREB211 gene was cloned using the reverse transcription PCR method.Its open reading frame（ORF）was 792 bp in length,encoding 263 amino acids,and containing the AP2 conserved domain unique to the AP2 family.Analysis of the upstream 2 kb sequence of the gene promoter revealed that it contains cis-acting elements related to growth and development,stress,hormone response,light response,and other functions.（2）The tissue differential expression analysis of the ZmEREB211 gene was per-formed using q RT-PCR technology.The results showed that the ZmEREB211 gene was mainly expressed in maize roots,with the highest expression level in young roots.Stress expression analysis showed that ZmEREB211 responded to mannitol,dehydration,Na Cl,cold stress,and hormone treatment to varying degrees.This indicates that the ZmEREB211 gene may play an important regulatory role in the growth and development of maize roots and their stress response.（3）The subcellular localization of ZmEREB211 fusion protein was performed through transient expression in tobacco,and the results showed that the green fluores-cence signal of ZmEREB211 fusion protein was mainly concentrated in the nucleus,in-dicating that ZmEREB211 protein was localized in the nucleus.（4）On 1/2 MS medium containing 150 mmol L-1 Na Cl,200 mmol L-1 mannitol,and100μmol L-1 JA,the main root length of transgenic Arabidopsis lines was significantly longer than that of wild type.It is speculated that the ZmEREB211 gene has a certain regulatory effect on salt and osmotic stress,and can respond to JA treatment.Under drought and high salt treatments in the soil,the growth status of transgenic Arabidopsis plants is better than that of wild-type Arabidopsis.After stress treatment,transgenic Ara-bidopsis plants have a higher green leaf rate,POD activity,and chlorophyll content,indi-cating that ZmEREB211 is a positive stress response factor in high salt,drought,and os-motic stress.（5）To further investigate the mechanism of ZmEREB211 gene involvement in drought and salt tolerance,q RT-PCR technology was used to analyze the expression of stress-responsive marker genes in transgenic Arabidopsis.The results showed that the expression levels of marker genes RD29A,RD29B,RAB18,and RD22 were significantly higher in transgenic plants than in wild-type plants,further demonstrating the important role of ZmEREB211 in resisting stress.（6）The results of the yeast two-hybrid experiment showed that ZmEREB211 inter-acted with ZmPP2C and ZmMYC7,respectively.Therefore,it is speculated that ZmEREB211 may jointly regulate the drought and salt tolerance process of plants through its interaction with ZmPP2C and ZmMYC7. |
