The Molecular Mechanism Of NAC Transcription Factor From Betula Platyphylla In Formation Of Secondary Wall And Abiotic Stress Response

NAC is one of the largest members of transcription factors,which is widely distributed from the moss plant to dicots.NAC transcription factors regulate the secondary metabolites of plants,respond to hormones and environmental factors,and involve in endogenous signaling.In this study,we cloned 24 BpNAC genes,which belong to the NAC transcription factor family from BetulaPlatyphylla transcriptoms,and the sequence characteristics of these 24BpNAC genes were analyzed.Then,we performed RT-PCR using different plant materials to identify the expression characteristics of these genes.The result indicated that BpNAC genes had different exression patterns among tissues or development stages.Based on the PCR results and sequence characteristics analysis,BpNAC5 was chosen for the further experiments,to understand its functions in secondary cell wall development and response to stress.The BpNAC5 protein was fused with GFP,and expressed in the onion epidermal cells by particle bombardment gun.The result showed that BpNAC5 is a nuclear protein.Then the promoter of BpNAC5 was analyzed using bioinformatics method,and some cis-acting elements were found,which response environmental stress.Thus,we deduced that the BpNAC5 was involved in the stress response regulatory network.The GUS was expressed driven by the BpNAC5 promoter in stems,leaves,and shoots of birch through transient expression assay.In all of these three organisms,the GUS expression activity were detected,and stems were stained most deeply.This results suggested the BpNAC5 promoter can drive the reporter gene expression,and the activity of BpNAC5 promoter is associated with the secondary xylem development of plants.The pROKII-BpNAC5 overexpression vector was transformated into Arabidopsis using the Agrobacterium tumefaciens-mediated method.The stage of stem development in the transgenic Arabidopsis came later than the wild type Arabidop.sis.However,the transgenic Arabidopsis lines were higher than wild type lines after the flower.Cell wall components analysis showed that cellulose content of the overexpression lines was significantly increased than the content of the wild type lines;there is no obvious differences of lignin content between the two lines;and hemicellulose content of transgenic Arabidopsis was decreased than that of the wild type Arabidopsis.The bases of the stem in transgenic and wild type Arabidopsis were examined in cross sections stained tests using phloroglucinol-HCl and calcofluor white.The results showed that more cellulose of the secondary wall components in overexpression lines than the wild type Arabidopsis,and the overexpression lines have larger lumens than the wild type lines.All these results suggested that BpNAC5is involved in the regulation of secondary cell wall synthesis,andhas a positive effect on cellulose synthesis.The overexpression of BpNAC5 and wild type Arabidopsis were treated under NaCl,ABA and mannitolstress.The results showed that the germination rate of the transgenic plants is higher than that of the wild type plants.The staining results using DAB,physiological NBT and Evans Blue showed that BpNAC5 gene can enhance the ability for resisting to environmental stress by scavenging reactive oxygen species in birch,which can cause cell damage.We also compared the physiological indicators,including the activities of SOD,POD,MDA,relative conductivity and chlorophyll content,between overexpression lines and wild type plants.The results showed BpNAC5 overexpression Arabidopsis has more capable to resist to the abiotic stress than wild type Arabidopsis.The BpNAC5 can recognize five cis-acting elements,including ACGTATERD1,CGACGOSAMY3,DOFCOREZM,,LTRECOREATCOR15 and TAAAGSTKST,which were detectd by yeast one-hybrid.Then the yeast one-hybrid system and transient expression assay,combing with bioinformatics analysis using PLACE website were carried out to identify the functions of these elements and the interaction between BpNAC5 and this five cis-acting elements.The result suggested that BpNAC5 can recognize these elements which are involved in the dehydration response,carbon metabolism,cold response and stomachange process.