Maize Genome-wide Prediction And Verification Of Target Genes Of MYB Transcription Factor

Transcriptional regulation of stress response gene expression is a crucial part of plant defense biotic and Abitoic stresses. As one of the largest plant transcription factor families, MYB transcription factors play an important role in plant stress tolerance. P1, C1, MYB-IF25genes in maize genome have been cloned, and verified that their encoded proteins have activity of transcription factor via abiotic stress-inducible. However, the MYB target genes and the specific signal transduction pathways is available for reference, but very limited. So far, the identified MYB target genes is rd22. Predicting MYB target genes on genome scale is helpful understanding the MYB regulation of resilience-related gene expression in specific circumstances, and resolving the molecular mechanisms of plant stress response.MYB protein in plant have a conserved R2R3DNA binding domain specifically recognized as the MYB binding elements on the combination of stress-responded gene promoter sequences, which have a core sequence TAACTG, to activate the expression of downstream target genes. In addition, identifying and binding to the target gene promoters is mutual synergy by multiple transcription factors, then initiate gene expression. Therefore, we can scan the whole maize genome to discover new MYB target genes by importing machine learning, one of the bioinformatics methods, due to the conserved core and flanking sequences of TFBS.In this study, we first modified the transcription factor binding sites (TFBS) of reported MYB target genes with HexDIFF algorithms, and constructed a support vector machine (SVM) classification model. Then we downloaded the maize inbred lines B73genome sequence and WGS gene annotation files from the Maize genome database (www.maizesequence.org). Homemade PERL scripts were used to get all annotated genes’ promoter, which are3000bp region of5’flanking sequences from transcription start site. Then, SVM classification models were used to scan these promoter sequences. The results showed435MYB binding sites and424downstream target genes. Via Gramene Mart, we exported1471Gene Ontology terms of229predicted target genes. GO enrichment analysis revealed that33predicted target genes (14.4%) which participate "response to stress" process, and40.6%located in the intracellular organelles. The main function of predicted target genes is adopted to plant stress tolerance, thus provide some support for the accuracy of our prediction method.In order to verify the predicted results, we first in vitro chemical synthesis the cDNA sequence of MYB-IF25, and built the prokaryotic expression vector, induced expression, separation of purification of the MYB-IF25combined protein. Then we randomly selected30predicted binding sites, to perform EMSA with MYB-IF25combined protein. The results showed that90%probes could bind to MYB-IF25combined protein in vitro, indicating that our strategy of predicting MYB binding site is credible. The results may provide a reference to the molecular mechanism of abiotic stress.