The Molecular Mechanism Of BplMYB46 From Betula Platyphylla In Mediating Drought And Salt Tolerance And Formation Of Secondary Wall

MYB (avian myeloblastosis) is one of the largest members of transcription factors. MYB transcription factors regulate the secondary metabolites of plants and respond to hormones and environmental factors. In this study, BplMYB46 transcription factor was found in transcriptome database of birch, which belongs to R2R3 MYB transcription factor and may be associated with the response to stress and formation of secondary wall in plant. In this experiment, function and molecular mechanism of BplMYB46 gene were mainly studied in response to abiotic stress and deposition of secondary cell wall. This research laid theoretical basis for birch under response to stress and regulation secondary metabolism.1. The BplMYB46 was fused with GFP driven by the CaMV 35S promoter, and introduced into onion epidermal cells by particle bombardment, the results showed that BplMYB46 is a nuclear protein.-The expression of BplMYB46 was up-regulated at 6-24h under NaCl, ABA and mannitol stress compared with untreated birch, this indicated that the expression of BplMYB46 could be induced by stress. The expression of BplMYB46 was highest in stems than leaves, a little higher in roots than leaves, which in top, middle and bottom from stems have increasing trend.2. In this research, pROK2-BplMYB46 overexpression vector and pFGC5941-promoter suppress expression vector was respectively transformated into birch by agrobacterium tumefaciens-mediated method, the birch of BplMYB46 overexpression, suppression expression transgennic and WT were treated under NaCl, ABA and mannitol stress. Results showed that birch of BplMYB46 overexpression had higher fresh weight, root length and content of chlorophyll, lower electrolyte leakage and water loss rate than those of WT under stress, BplMYB46 can enhance activities of SOD, POD and content of praline by regulating the espression of SOD and POD, P5CS, P5CDH and ProDH reduced the accumulation of ROS. These results demonstrated that BplMYB46 can enhance resistance of birch to stress.3. The bases of the stem in transgenic and WT birch were examined in cross sections stained using toluidine blue, phloroglucinol-HCl and calcofluor white, the results suggested that the more deposition of secondary wall components, including lignin and cellulose in the overexpressors than wild-type birch, the thinnest in the repressions. Contents of lignin and hemicellulose of BplMYB46 overexpression birch were higher than those of WT, content of cellulose was lower than those of WT, in contrast to suppression of BplMYB46. The expression of PAL, CCo,4CL, POD, LAC and Cesa related to synthesis of lignin and cellulose and MYBs possible related to synthesis of secondary cell wall were up-regulated in BplMYB46 overexpression birch, down-regulated in suppression of BplMYB46 when WT as control. These results demonstrated that BplMYB46 can promote deposition of secondary cell wall by regulating the expression of enzymes and transcription factors related to synthesis of secondary cell wall.4. BplMYB46 has transcriptional activation activity, the region from 697 to 915bp of BplMYB46 was also found to have transactivation activity using yeast two-hybrid, and BplMYB46 can form homodimers with itself, and five out of nine MYBs could interact with the BplMYB46 protein to form heterodimers.5. The BplMYB46 can recognise 8 elements by yeast one-hybrid, GUS staining and activity:E-box was associated with light-responsive and biosynthesis genes of phenylpropanoid, LTRECORE was critical to the low-temperature response, GT-1 can strengthen resistance to the stress of pathogen and salt in plants, M1 was MBSI element involved in cold and drought stresses, M2 was an element related to biosynthesis of secondary cell wall identified by AtMYB46, M3 was tolarance to drought. TCbox and AGbox were two unknown elements, which may be new elements recognized by BplMYB46. BplMYB46 can regulate the expression of target genes related to mediating abiotic stress tolerance and formation of secondary wall by binding to cis-acting elements of their promoter using CHIP-PCR technology.6. The results of DGE indicated that there are 2421 significantly expressed genes between overexpression and suppress expression birch, including 952 up-regulation genes and 1469 down-regulation genes. The differential genes were mainly involved in the metabolic pathways, biosynthesis of secondary metabolites, plant hormone signal transduction pathways, phenylpropanoid and flavonoid biosynthesis. There are some ERF, SOD, POD, HSP genes related to resistance the stress of birch, CAD, CCR and LAC genes associated with lignin biosynthesis, and cellulose synthase genes. The results indicated that BplMYB46 gene can regulate the expression of genes involved in tolerant to stress and deposition of secondary cell wall.