BpSPL8 Transcription Factor Transformation And Functional Verification Of Betula Platyphylla Suk.

SPLs are a class of transcription factors that bind to the SQUAMOSA promoter and play an important role in plant growth and development.In the previous study,we discovered a non-miR156-targeted BpSPL8 gene from Betula platyphylla Suk.,which can affect the development of leaves and roots of transgenic plants after transformation into Arabidopsis.This paper will further study the mechanism of BpSPL8 regulation of plant development.A promoter fragment of 2028 bp upstream the BpSPL8 initiation codon was isolated from Betula platyphylla by cloning.The plant expression vectors with GUS(?-glucuronidase coding gene)expression driven by the promoters of BpSPL8 was constructed and transformed into Arabidopsis thaliana by the floral dip method.Through the detection of GUS activity,the tissue expression pattern of the BpSPL8 promoter in Arabidopsis was analyzed.Promoter element analysis revealed that BpSPL8 promoter contains elements for tissue-specific expression,light-responsive,hormone-responsive and stress-responsive.GUS histochemical staining results showed that GUS activity was observed in hypocotyls,leaves,petioles,roots and inflorescences of transgenic Arabidopsis carrying the BpSPL8 promoter.BpSPL8 may affect the development of leaves,petioles,roots and inflorescences of Betula platyphylla,and participate the response to light,hormone and stress.The expression patterns of BpSPL8 gene in roots and leaves of birch were up-regulated and then down-regulated under PEG treatment.Finally,the overexpression of BpSPL8 Arabidopsis thaliana was used to explore the function of BpSPL8 in the drought process.Drought stress tolerance pointed out that the transgenic plants showed significantly lower survival rate and proline content than wild type,while malondialdehyde content was higher than wild type.Two known stress-resistant genes,DR29B and P5CS1 showed delayed up-regulation in transgenic Arabidopsis.Ectopic overexpression of BpSPL8 can reduce the drought tolerance Arabidopsis thaliana and affect the expression patterns of resistance genes DR29B and P5CS1 under drought stress.BpSPL8 was transformed into Betula platyphylla by Agrobacterium-mediated leaf disc method;the transgenic white birch phenotype was analyzed by comparison with the wild type.Overexpression of BpSPL8 resulted in a slower initial rate and a decrease in the number of birch roots,and also affected total roots length.After 30 days of transplanting,the stems and petioles of white birch appeared green and the wild type showed reddish brown.qRT-PCR detected the expression level of BpCHS gene in transgenic and wild type results showed that the three BpCHS genes of birch were strongly inhibited in BpSPL8 overexpressing lines.After 10 days of drought,the wild-type leaves grew normally and the transgenic birch leaves showed wilting.The plant height statistics of Betula platyphylla showed that the transgenic birch plant height was significantly lower than that of the wild type.The above results indicated that BpSPL8 plays a multifunctional regulation in the growth and development of Betula platyphylla,including the growth and development of white birch roots and stems;negatively regulating the drought tolerance of Betula platyphylla;inhibiting the expression of BpCHS gene affects the synthesis of flavonoids,thereby reducing the pigment in the stem segment and accumulation in the petiole.