Bioinformatics Analysis, Cloning And Expression Of MYB Transcription Factor Genes In Sugarcane

Transcription factor is trans-acting factor, is a type of DNA binding protein which can combine specifically with the cis-acting element, silencer or enhancer in gene promoter region and interact with these elements to activate or suppress the transcription and translation of target genes. Previous studies showed that the plant MYB transcription factor acts an important role in response to adversity stress, but very limited reports on sugarcane MYB transcription factors were found. The research on sugarcane MYB, supported by the funds of the National "948" project (2010-C21) and the state system of modern industrial technology projects, was carried out. In this study, twenty-one electronic cloning of sugarcane MYB transcription factor family genes were gained by making use of sugarcane EST databases, gene databases and protein databases. And then, three MYB genes were selected out for informatics analysis in details and the experimental cloning was conducted. In order to identification of the function of MYB18, the expression of the MYB18under the modified stress in sugarcane detected by Real-time RT-PCR and the prokaryotic expression in E. coli were studied. The results provided the primary understand for the gene function and the basis for dinning the function gene for the improvement of resistance in sugarcane. Thus, the research has an important practical value and the theoretical significance. The main results and the conclusions for this study are as follows,1. In this study, twenty-one electronic cloning of sugarcane MYB transcription factor family genes were gained by making use of sugarcane EST databases, gene databases and protein databases. Bioinformatics analysis showed the typical domain of MYB family being observed in the sequences of these genes,17with the R2R3domain, only one with the R3domain and the other three being unclear. Three transcription factor genes termed as MYB2, MYB]8and MYB59in sugarcane were selected for detail sequence-based prediction of the hydrophobic, isoelectric point, secondary and tertiary structure.2. RT-PCR was carried out for cloning the cDNA sequences of MYB18and MYB59transcription factor genes. The sequences are same to the sequences obtained in silico. By means of multiple comparision the nucleic acid sequences of the above two genes with those in several other relative plant species, and then the evolutionary tree was formed. High homologous was observed when comparison the two sugarcane MYB transcription factors MYB18and MYB59with the MYB genes isolated from maize, one of the close relative species of sugarcane, In order to understand the structures of the two MYB genes, DNA sequences of MYB18and MYB59transcription factor genes were isolated from genome DNA of sugarcane. By comparison the corresponding cDNA sequences with the DNA sequences of MYB18and MYB59genes, the exon and intron structure were revealed. Clearly, both of the genes contain two introns,116bp and92bp for MYB18gene and97bp and88bp for MYB59gene, respectively. The results provide the basis for the further functional analysis.When investigation the synonymous codon usage in maize, sorghum and rice MYB family transcription factor genes, We found highly conserved in protein sequence and similarity in codon usage for MYB transcription factor among the three different but related plant species thougt some codon usage being different. For example, considering the codon usage for valine (Val), corn and sorghum prefer to the codons of GUC and GUG respectively, but rice prefers to GUC and GUG. When considering the codon usage for threonine (Thr), rice prefers to ACG, but sorghum and maize share same preference to ACG and ACC. In addition, the bias of codon usage was affected by base composition of the gene in some degree.4. In order to identify the function of MYB18, both the protein expression in prokaryote for MYB18and the MYB18gene expression in sugarcane root tissues detected by quantitative RT-PCR under the modified adversity stress were carried out. The results of quantitative PCR expression analysis showed that MYB18expression in sugarcane was strong inhibited by the stress of NaCl, H2O2and PEG. It indicated that MYB18in sugarcane may play some roles in response to abiotic stress of salt and drough and to biotic stress. In addition, the specific protein expression in the E. coli strain of Rosetta (DE3) with the vector of pET28was achieved under the induction of IPTG, but not in the mock and in negative strain without MYB18. The above results suggest MYBl8gene may act an important role in the defense response. The purified MYB18protein was obtained by the meaning of His labels. The results provide the basis for further study on gene regulatory networks and lay the foundation for the genetically modification of sugarcane.