Evaluation Of Target Tenes Of Resistant Transcription Factor MYB In Maize

Transcription factors recognize and bind to the highly conserved specific nucleotide sequences at the upstream promoter regions of its target genes, and regulate temporal and spatial expression of its targe genes. Prediction and evaluation of target genes of transcription factors is an important method for functional genomics research. It provides reference for the gene function resolution, and help to understand the regulatory mechanisms of gene expression and construct regulation network of gene expression.MYB transcription factor, a plant ortholog of myeloblastosis (MYB) transcription factor in human, plays a very important role in transcriptional regulation included in plant metabolism, stress response and many other grwoth and development processes. Studies have shown that the specific core sequence that maize MYB recognizes is hexamer "TAACTG", where the third base A is highly conserved, and plays a pivotal role in the recognition of MYB target genes. In the model plants Arabidopsis and rice, some literatures are available for prediction and evaluation of MYB target genes. In maize, only rd22, CAB, Bzl and Bz2 were evaluated to be the target genes of MYB. In this study, the target genes of MYB were predicted genome-wide, their upstream promoters were cloned and evaluated for their promotion activities by transient expression in maize, in order to provide reference for resilience mechanism and regulatory network of MYB transcription factor.On the basis of the previous studies, the promoters of 136,770 annotated genes were firstly obtained in maize genome by using software Promoter 2.0. Then,369 possible binding sequence of MYB transription factor were predicted from the promotors of the 136,770 annotated genes by a support vector machine (SVM) classifier constructed using a Perl language script to combine HexDIFF algorithm with library support vector machine (Lib-SVM). The downstrem encoding sequences were predicted as the target genes of the MYB transcription factor.2,575 terms of Ontology annotations were exported for 181 of these target genes by GrameneMart, and enriched using AgriGO. The result showed that 63 of the 181 target genes were involved in stress response.Five encoding sequences (GRMZM2G030181, GRMZM2G081622, GRMZM2G056365, GRMZM2G115698 and GRMZM2G310161) were selected randomly from the Gramene database, and the upstream promoter sequences were cloned and, used to replace with the CaMV35S promoter at the upstream of the GUS gene in plasmid pBI221, to construct wild-type monocot transient expression vectors. Then, the core sequences TAACTG was mutated by replacing the third A base with G base by piecewise PCR amplification, and the mutated promoter sequences were inserted at the same positions of the pBI221 plasmid to replace the CaMV35S promoter at the upstream of the GUS gene and construct five mutated expression vectors. Maize calli were transformed with a total of the ten wild-type and mutant expression vectors by particle bombardment, cultured on hypertonic medium with 8% mannitol at 27℃ in dark for 24 h. The result of histochemical staining showed that all the wild-type and mutant promoters of the five genes drived the GUS gene expressing in maize calli under the hypertonic stress condition. The ratio of the GUS activity relative to the luciferase activity (internal control) (GUS/LUC) was measured, and used to evaluate the promotion activity of the wild-type and the mutant promoter sequences under the hypertonic stress condition. The results showed that the promotion activities of the wild-type promoter sequences of the encoding sequences GRMZM2G030181, GRMZM2G115698 and GRMZM2G31016 were significantly higher than that of their mutated sequences. The promotion activity of the wild-type promoter sequence of the encoding sequence GRMZM2G081622 was greatly significantly higher than that of its mutated sequence. However, the promotion activity of the wile-type promoter sequence of the encoding sequence GRMZM2G056365 was non-significant different from that of its mutated sequence, indicating that this promoter sequence was not regulated strongly by the MYB transcription factor.In conclusion, the strategy we developed is feasible for the prediction of the regulated promoter sequences and target genes of transcription factors, and is useful for effect analysis of transcription factors on regulation network of gene expression, to provide reference for functional genomics research.