Study On Molecular Mechanisms Of Two MYB Transcription Factor Genes In Canola And Arabidopsis Regulating ROS And Jasmonate Signal Transductions,respectively

As one of the largest transcription factor families,MYB(myeloblastosis)transcription factor family plays important roles in the whole process of plant growth and development.They also regulate the secondary metabolic processes,such as phenylpropanoid pathway.Meanwhile,they also participate in plant response to biotic and abiotic stresses,and at the same time,some signaling transduction pathways are also implicated in these processes to form a complicated and important regulatory network.Although,much research about MYB function in Arabidopsis has been reported,the functions of many MYB genes are still unknown.In addation,in the oilseed rape or canola(Brassica napus L)belonging to cruciferae as arabidopsis,only a few studies about the MYB functions have been reported.It is therfore necessary to study the functions and molecular mechanis of MYB genes in both arabidopsis and canola.In a recent study,on the base of identification,cDNA cloning,transcriptional activity detection,subcellular localization analysis and functional screening of R3R3-MYB transcription factors in canola,we studied the function and the regulatory molecular mechanism of Bna MYB78 systematically.Transient expression of BnaMYB78 in the tobacco led to accumulation of reactive oxygen species(ROS)and cell death.We confirmed this function through DAB staining,quantitative determination of chlorophyll,malondialdehyde(MDA),hydrogenperoxide(H2O2)and so on.The result of qRT-PCR revealed that the relative expression level of BnaMYB78 in senescent leaf was increased.BnaMYB78 is a trans-activator protein and located in nuclei.Then we found that Rboh B,PR2,PR5,GST,ACRE31,HIN1 may be the downstream targets of BnaMYB78 through qRT-PCR screening,and verified that BnaMYB78 could regulate the GST and HIN1 by dual-LUC assay.Besides,BnaMYB78 was identified to regulate the expression of fLUC driven by three different MYB cis-elements.During our research we identified a R2R3-MYB gene AtMYBx in Arabidopsis and its overexpression lines showed senescence earlier than the control line under both dark and natural conditions.The result of qRT-PCR indicated that JA could increase the relative expression of AtMYBx.AtMYBx is a transactivator protein and located in the nucleus.AtMYBx could bind to three cis-elements through dual-LUC and electrophoretic mobility shift assay(EMSA)assays.Then we detected the changes at the mRNA levels of JA-and senescence-related genes in AtMYBx-related lines and found that expression levels of some JA-related genes changed significantly in the AtMYBx overexpression lines,which was partially confirmed by the dual-LUC assay.Meanwhile the result of quantitative determination of JA contents in AtMYBx-related lines revealed that the synthesis and accumulation of JA increased significantly in AtMYBx overexpression lines.All the results indicated that AtMYBx could activate the transcription of LOX2 and increase the JA synthesis and accumulation,and the high level of JA induces earlier senescence.Our research not only discovered two new R2R3-MYB genes which regulate the ROS and JA signal transductions respectively,but also identified their expression patterns,transcriptional activities,and analyzed their target genes and molecular mechanisms preliminarily.This work will lay a foundation for studying the biological functions and regulatory molecular mechanism.