Function Analysis Of BpSPL6 And BpSPL2 In Adventitious Root Formation In Betula Platyphylla Suk.

SQUAMOSA-promoter binding protein-like(SPL)proteins are plant-specific transcript factors that play essential roles in regulating various developmental processes,including plant phase transition,flower development,sporogenesis,leaf and root development and stress response.This paper will study the function of BpSPL6 and BpSPL2 genes in G1 group of Betula platyphylla Suk.(birch)SPL gene family in adventitious root formation.A 1703 bp promoter sequence of BpSPL6 gene was cloned from B.platyphylla genomic DNA.Sequence analysis showed that the BpSPL6 gene promoter included cis-element for specific parts(root pollen),hormone response elements(Auxin,Gibberellin,Salicylic acid,Abscisic acid)and dehydration response elements.The GUS staining results of transgenic A.thaliana showed that the GUS gene driven by BpSPL6 gene promoter was mainly expressed in leaves and roots.When transgenic A.thaliana was subjected to sodium chloride and mannitol stress,the expression level of GUS gene driven by BpSPL6 gene promoter decreased compared with the control.These suggesting that BpSPL6 gene may be involved in plant leaf and root development and response to salt and drought stress.And the BpSPL6 gene of ORF sequences was cloned and the BpSPL6 gene overexpression vectors(pBI121-BpSPL6-GFP)and inhibition expression vectors(pROK Ⅱ-BpSPL6-SRDX)were constructed.Subcellular localization found BpSPL6 gene localized in the nucleus and cytoplasm.BpSPL6 gene overexpression and inhibition expression of transgenic lines were successfully obtained by leaf disc transformation.Overexpression of BpSPL6 gene significantly decreased the number of adventitious roots,and significantly increased the number of adventitious roots in pROK Ⅱ-BpSPL6-SRDX birch.BpSPL2 gene overexpression and inhibition expression of birch seedlings were cultured,and the expression of related genes in adventitious root development was analysed by qRT-PCR.BpSPL2 gene down-regulated the expression of ASA1 and NAC1 genes that are positive regulators of auxin signaling,and IAMT1 gene that is a negative regulator of auxin signaling,and promotes NINJA gene that is a negative regulator of jasmonic acid signaling.Treatment gibberellin and its inhibitor(PBZ)in WT birch seedlings showed that gibberellin negatively regulated the number of adventitious roots.The number of adventitious roots decreased after gibberellin treatment,the number of adventitious roots increased after PBZ treatment,and gibberellin and its inhibitors could partially restore the phenotype caused by BpSPL2 gene.The expression of PIF4 gene that negatively regulating gibberellin signaling was inhibited by BpSPL2 gene.Therefore,BpSPL2 gene may inhibits the expression of PIF4 gene,thereby promoting the accumulation of gibberellin and inhibiting the formation of adventitious roots of birch.Moreover,the expression of three possible BpSPL2 target genes was analysed by qRT-PCR,and it was found that CYP71A25、LAC14 and CSLE1 genes showed opposite expression patterns in BpSPL2 overexpression and inhibition expression birch,so they may be the target genes of BpSPL2 gene.