Functional Analysis Of AP2/ERF Transcription Factor SlERF.F4 In Fruit Ripening And Response To Drought Stress

Tomato（Solanum lycopersicum）,rich in vitamins,lycopene and other nutrients beneficial to the human body,and is one of the important vegetable crops in the world.Due to its small genome size,clear genetic background,easy transformation,available mutants,short growth cycle,and with fruit,tomato is considered as a model system for studying fruit development and adversity stress responses.ERF（Ethylene Responsive Factor）transcription factors belong to the AP2/ERF family,which play an important role in plant growth and development and various stress responses.In this study,we isolated SlERF.F4 from Micro Tom,conducted bioinformatics analysis determined its expression level in different organ of the plants and the response to exogenous hormones and abiotic stresses.We also investigated the subcellular localization of SlERF.F4 by protoplast transient transfection method.We constructed SlERF.F4 overexpressing plasmid and obtained SlERF.F4 transgenic tomato plants by Agrobacterium tumefaciens method.We also analyzed the function of SlERF.F4infruit ripening and its response to drought stress.The main results of this study are as follows:（1）We first carried out bioinformatics methods to analyze the gene structure of SlERF.F4.There was 678 bp in nucleotide sequence,encoding 225 amino acids.Promoter components analysis showed that there are many cis-elements related to light response and hormone response.The results of phylogenetic trees showed that SlERF3 had high homology with SlERF.F4.（2）We also determined SlERF.F4 expression pattern.The result showed that SlERF.F4 had high expression level in flower and fruit of tomato,indicating that SlERF.F4 may regulate flower and fruit development.It was found that the expression of SlERF.F4 gene was induced by dehydration,salt,low and high temperature treatments.For plant hormone treatment,SlERF.F4 was highly induced by ABA and BR,while inhibited at 6 h and induced at 24 h after SA treatment.Above results suggested that the expression of SlERF.F4 gene is regulated by abiotic stresses and plant hormones at the transcription level.（3）Overexpression vector pCambia1300-SlERF.F4-YFP was constructed and transformed into tomato mediated by Agrobacterium tumefaciens.Four positive T₀generation plants were obtained after hygromycin selection.Homozygous T₂generation plants were obtained and used for further study.The expression level of SlERF.F4 gene was about 2 folds to WT in two SlERF.F4 transgenic lines,respectively.After observing the phenotype,it was found that the transgenic plants exhibited shorter plant height,reduced fruit number,lower yield,and the delayed fruit ripening.Furthermore,the expression of ripening-related genes,including RIN,TAG,CNR,E4,E8 and PG were reduced in SlERF.F4-overexpressing lines at different ripening stages including MG（Mature Green）,Br（Breaker）,Br+4（4d after Br）and Br+7（7d after Br）.（4）We also studied the response of SlERF.F4 transgenic plants and wild-type to drought stress,and the results showed that the wilting degree of transgenic plants was lighter and had a higher survival rate under drought conditions.The results indicate that SlERF.F4 can effectively improve drought resistance in transgenic plants under drought conditions.The above results indicate that SlERF.F4 plays a certain function in tomato fruit ripening and response to drought stress,which provides a theoretical basis for improving tomato quality and drought resistance by genetic engineering.