Study On The Upstream Regulatory Mechanism Of BplMYB46 Gene In Betula Platyphyll

In plants,trans-acting factors(also known as transcription factors)regulate the expressions of target genes via specifically binding to cis-acting elements on the promoters of downstream genes,thus affecting the growth and development of plants.In previous research,the BplMYB46 gene from birch could enhance the tolerance to salt and osmotic stresses,participate in secondary wall synthesis.The downstream regulation mechanism of the BplMYB46 gene was clarified,however,its upstream regulatory mechanism has not been uncovered.In this study,we analyzed the upstream regulation mechanism of the BplMYB46 gene,thus can enrich the mechanism of the BplMYB46 gene regulating the salt and osmotic tolerances in birch,and obtained its upstream regulatory factors via screening,can discover new high-quality genes controlling plant growth and development,and also can provide the theoretical and practical bases for the breeding of new varieties of forest trees using molecular biology methods in the future.The cis-acting elements of the BplMYB46 promoter were predicted,using the PLACE online software.The result found the Dof,MYC,ABRE,W-box,and so on related to photosynthesis pathway and response to various stresses on the promoter.The constructed fused of 35s-BplMYB46 promoter with GUS gene was transferred to Nicotiana benthamiana using acupuncture transient transformation method,and the relative GUS activities were detected under NaCl and Mannitol treatments.The results showed that the relative GUS activities driven by the BplMYB46 promoter were obviously increased compared with control,indicating that the promoter of BplMYB46 could respond to high salt and osmotic stress.The birch cDNA library was constructed,and eight upstream regulatory factors were screened using the yeast one-hybrid system,including BplDof1,BplDof7,BplDof10,BplDof12,BplbZIP3,BplbZIP4,BplbZIP5 and BplWRKY3,and they could specifically bind to the Dof,ABRE and W-box cis-acting elements on the BplMYB46 promoter.After constructing effectors and reporter vectors,the yeast one-hybrid and qRT-PCR results indicated that the eight upstream regulatory factors could bind to specific cis-acting element,respectively,and regulate the expression of BplMYB46 gene through their bindings.The cDNAs from the different birch tissues under high salt and osmotic stresses were used as templates,and qRT-PCR was used to analyze the expression changes of the eight regulatory factors.The results showed that the expressions of the BplDof1,BplDof 7,BplDof10,BplDof12,BplbZIP3,BplbZIP4,BplbZIP5,and BplWRKY3 can be induced under high salt and osmotic stress.Further,the fused construct of the regulatory gene with GFP was introduced into onion epidermal cells by particle bombardment,respectively,and the subcellular localizations of the eight regulatory factors were completed.The results showed that they all located in the nucleus.