Transcription Regulation Of Egg Cell-specific Expression Gene EGG1 In Arabidopsis

In the process of double fertilization,the egg cell is combined with a sperm cell to develop into an embryo,and the central cell is combined with another sperm to develop into an endosperm,which finally completes the entire process of double fertilization.As the female gamete,the egg cell not only participates in the fertilization process,but also has intercellular communication with the member cells of the embryo sac to coordinate each other’s cell fates and developmental process.Transcriptome data showed that some genes were specifically expressed in the egg cell,and a few genes were reported that they were important for the function of the egg cell.The mechanisms that control the expression of genes in eggs are not clear.In the previous work of our laboratory,it was found that EGG1,an egg cell-specific expression gene,was involved in the intercellular communication of double fertilization,which ensured the normal progress of double fertilization.However,the transcriptional regulation mechanism of EGG1 gene is still unclear.In this paper,the transcriptional activity of EGG1 promoters of different lengths was analyzed to find important regulatory elements.Using yeast one-hybrid technology,we found some transcription factors interacting with the EGG1 promoter.The binding sites analysis of the selected transcription factors provide information for revealing the transcriptional regulation mechanism of EGG1 gene.The main experimental results were summarized as following:1.Analysis of the expression pattern of EGG1 gene: In order to explore the expression of this gene in the female gametophyte,we constructed the pEGG1::H2B-EGFP expression vector,and the results showed that EGG1 is an egg cell-specific expression gene.2.The functional region of the EGG1 gene promoter: To explore the important regulatory elements of EGG1 gene promoter,500 bp,350bp,200 bp and 100 bp promoter fragments are cloned into an expression vector with H2B-GFP as a reporter gene.The results show that the regulatory elements that drive the specific expression of the EGG1 gene are located between 100bp-200 bp upstream of the promoter.3.Yeast one-hybrid screening of transcription factors: Yeast one-hybrid screening was usedto identify transcription factors binding to the EGG1 promoter.Four candidate transcription factors that may activate EGG1 are obtained.Through further interaction verification,it was finally confirmed that the transcription factors bZIP11 and bZIP44 bind to the EGG1 promoter and participate in the regulation of EGG1 gene expression.4.Transcription factor and EGG1 promoter binding analysis: By online analyzing we got the predicted binding sites of the EGG1 promoter and bZIP11 and bZIP44.We constructed the expression vectors of base mutation and base deletion at the prediction site and transformed plant with these vectors.The results show that the mutant EGG1 promoter drive GFP expression not only in egg cell,but also in synergid,central cell and antipodal cell in transgenic plants.It indicates that the specific expression of EGG1 gene may need two mechanisms: one is to regulate the expression of EGG1 gene in egg cells,and the other is to regulate that EGG1 gene is not expressed in other cells.When the binding site is deleted,it does not affect the expression of the EGG1 gene in egg cells,indicating that the transcription factors bZIP11 and bZIP44 may bind to the EGG1 promoter to inhibit EGG1 expression in other cells.