| As one of the largest and functionally diverse transcription factor families in plant, the MYB family of proteins is widely involved in regulating phenylpropanoid metabolism, cell morphogenesis, cell cycle and responding to abiotic and biotic stresses. Phenylpropanoid metabolism is a significant secondary metabolic pathway, which can synthesize all phenylpropanoid compounds in plant directly or indirectly. Some evidences suggested that the phenylpropanoid metabolism evolved earlier than the rise of tracheophytes with a conserved evolution. Phenylpropanoid metabolism plays an important role in plant stress tolerance. Lignin as a natural barrier in plant cell wall, protected the plant against disease stress, provided a mechanical support and hydrophobic vascular system, increased the ability of plant resistance to lodging and drought. Anthocyanins had been certified were able to clear active oxygen and free radicals accumulated in plant, and protect plants from oxidative damage caused by adversity stress. The proanthocyanidins deposited in the endothelial layer of the seed coat in many species. The main function of proanthocyanidins were to reinforce the strength of seed coat, induce seeds to hibernate, provide protection against microbial pathogens. Flavonols as the Uv filters were able to absorb the ultraviolet lights, and avoid the photosynthetic organs in plant radiation by ultraviolet.The maize had been a paramount staple crop in China. Intensive study the phenylpropanoid metabolism in maize is important to breed highly resistance and quality maize varieties. Compared with Arabidopsis and other dicots species, the phenylpropanoid metabolism studied in maize and other monocots are significantly backward. R2R3MYB transcription factors have been widely demonstrated to rugulate phenylpropanoid metabolism in Arabidopsis and other dicots species, which provided important information for exploring the phenylpropanoid metabolism in maize. In the previous study, we performed a comprehensive analysis of the R2R3MYB family 158 geges in maize genomes, include the chromosomal distribution and evolution mechanism. Take advantage of the homology analysis, co-expression analysis and collinearity analysis, we got several conserved R2R3MYB transcription factors in regulating phenylpropanoid metabolism. In the present study, we cloned two R2R3MYB genes from maize inbred line B73, named ZmMYB111and ZmMYB148. Furthermore we explored the function of ZmMYB111and ZmMYB148 involved in regulating phenylpropanoid metabolism. The detail contents of this study are as follows:1. This study we successfully got the open reading frames of ZmMYB111and ZmMYB148 gene by PCR method. Analysis the amino acid sequence and senior structure of ZmMYB111and ZmMYB148 protein, those results indicated that ZmMYB111and ZmMYB148 were typical plant R2R3MYB transcription factor. The phylogenetic tree (NJ tree) was built with the homology protein of ZmMYB111 and ZmMYB148 from other species, the results demonstrated that ZmMYB111and ZmMYB148 shared closely phylogenetic relationship and contained the conservative motif with R2R3MYB from other species.2. Real Time PCR analysis indicated that ZmMYB111 was no expression in seeding leave, pith, female flower,the expression level in leave,seeding root,female flower is low, the expression level in root,endosperm,seed is medium,upper of the stem is high, bottom of the stem is the highest in all tissues. ZmMYB148 was no expression in seeding leave, pith, female flower, the expression level in leave and female flower is low, the expression level in seeding root, upper of the stem, seed is medium, the expression level in root, bottom of the stem is high, the expression level in endosperm is the highest in all tissues.3. Transactivation activity assays showed the ZmMYB111and ZmMYB148 protein contain transcriptional activation domain.4. Utilizing gege gun transformed construction plamid of ZmMYBllland ZmMYB148 into onion cells, the results indicated that ZmMYB148 was localized in the nucleus,ZmMYB111 was localized in the nucleus and cytoplasmic membrane.5. Cloned the promoter of phenylalnine ammonialyase and 4-coumaric acid CO A ligase, which are the key enzyme of phenylpropanoid metabolism,are used for the yeast one-hybrid experiment.The result showed that ZmMYB111, ZmMYB148 could combine with the promoter of phenylalnine ammonialyase and 4-coumaric acid COA ligase in vitro.6. PALis the rate-limiting enzyme of phenylpropanoid metabolism, in order to study the ZmMYBlll and ZmMYB148 can activate or inhibit the activity of the promoter of PAL, the co-transformation the ZmMYB111 and promoter of PAL or the ZmMYB148 and promoter of PAL into maize endosperm through Gene gun bombardment. The result demonstrated that ZmMYB111and ZmMYB148 could activite the pPAL, which is consistent with the combination between the transcription factor and promoter by the yeast one-hybrid experiment... |
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