Cloning And Transformation Of MYB Transcription Factors Based On Bambusa Emeiensis Transcriptome

Bambusa emeiensis Chia et H.L.Fung cv.Viridiflava belongs to Bambusoideae Neosinocalamus, which is good papermaking materials in southwest China. But the utilization of fiber in Bambusa emeiensis was limited by its high content of lignin, at the same time the bamboo was influenced by freezing, drought and so on, which caused massive decline in its production. It was reported that MYB transcription factors play an important role in regulation of plant secondary cell wall synthesis and in response to abiotic stresses. In this research, two MYB genes were cloned based on Bambusa emeiensis shoots transcriptome database. Their bioinformatics, tissue expression patterns, stress induced expression analysis and transformation in tobacco were performed. The studies will provide a theoretical basis for improving resistance and regulation of lignin biosynthesis in industrial bamboo by transgenic engineering. The main results are as follows:Two MYB sequences were selected from the transcriptome database. The full-length primers were designed and the cDNA from the shoots with 100 cm was choosed as template. Two MYB genes were cloned and named BeMYB1 and BeMYB2, respectively. Their accession numbers in GenBank were KJ496128 and KJ496129, respectively.Bioinformatics analysis showed that cDNA full sequence of BeMYB1 and BeMYB2 were 2061 bp and 1142 bp, respectively. The sequence length of cDNA were 1899 bp, encoding 632 amino acids and 1017 bp, encoding 338 amino acids, respectively. The proteins encoded by BeMYB1 and BeMYB2 genes were both hydrophilic, which had 10 and 12 phosphorylation sites, 13 and 5 glycosylation sites, respectively. Amino acid sequence analysis showed that protein encoded by BeMYB1 gene had a MYB domain in the C-terminal, which had a high similarity with wheat TaMYB48, they belonged to MYB-related protein. While the protein encoded by BeMYB2 had two MYB domains at the N-terminus, which belonged to R2R3-MYB protein subfamily, and it was clustered into one clade with the PeMYB2 and OsMYB18.The expression patterns in different tissues analysis of BeMYB1 and BeMYB2 genes showed that BeMYB1 and BeMYB2 were both expressed in shoot, leave and stem in Bambusa emeiensis, BeMYB1 mainly expressed in stem while BeMYB2 mainly expressed in leave.Bambusa emeiensis seedlings were treated with ABA(200 μmol ? L-1), NaCl(200 mmol ? L-1) and PEG 6000(20%) stress. The results of stress-induced differences expression analysis of BeMYB1 and BeMYB2 genes by Real-time PCR technology showed that BeMYB1 and BeMYB2 genes were both responding to ABA, high salt and drought stresses.The plant over-expressing vectors named pCAMBIA1303-BeMYB1 and pCAMBIA1303-BeMYB2 were successfully constructed. They were successfully transformed into tobacco. The transgenic tobacco plants were obtained. Preliminary results of the analysis of transgenic tobacco plants showed that there were delayed flowering, stout stems, higher plants and decreased lignin content in the transgenic tobacco with pCAMBIA1303- BeMYB1, when compared with CK plant. While the flowering period of transgenic tabacoo plants with pCAMBIA 1303- BeMYB2 were consistent with CK. The pCAMBIA2301- BeMYB1-RNAi and pCAMBIA 2301- BeMYB2-RNAi interference victors were also constructed to study the function of BeMYB1 and BeMYB2 in future time.