Cloning And Transformation Of BrMYB77 Transcription Factor Gene From Turnip 'Tsuda'

Transcription factors play very important roles in the regulation of plant growth, plant development and plant response to environment. MYB transcription factor gene is one of the largest family genes in plants, which involved in regulation of secondary metabolism responding to hormones, environmental factors, also regulation of cell differentiation, cell cycles and the formation of leaves and other organs. In this study, Brassica rapa'Tsuda', which has a characteristic of the light-dependent anthocyanin biosynthesis, was used as materials. One of MYB family genes was cloned using RT-PCR. The main results were obtained as follows.1 One of MYB family genes was cloned from Brassica rapa'Tsuda'. The homology between the sequence of fragment cloned of Brassica rapa 'Tsuda'and MYB77 of Arabidopsis thaliana is up to 54%, Comparison of deduced aminoacid sequences showed that it is a typical R2R3MYB transcription factors.2 The transient expression vector pA7-BrMYB77 consisting of GFP gene was constructed and its transient expression showed the nuclear localization of BrMYB77. The geen fluorescent protein expression vector was constructed and transformed to the epidermal cells of onion by vacuum infiltration method. The result of instantaneous expression showed that BrMYB77 was localized in cell nucleus of onion cell.3 The fragment of BrMYB77 coding sequence was ligated into expression vector pGEX-KG and was transformed into E.coli Rosetta (DE3) strain. Then the E.coli Rosetta (DE3) strain contained the recombinant vector was induced by IPTG to express fusion protein. GST-BrMYB77 fusion protein was obtained and verified by SDS-PAGE and Western blotting analysis. It provides the foundation to the study the function of BrMYB77.4 With Gateway clonging system, expression vector, pCAMBIA1301-PMI-BrMYB77 was constructed and transformed into tomato Solanum lycopericum 'Money Maker'with Agrobacterium EHA105. The positive tomato transformants were selected using MS medium containing mannose and was identified by PCR.