Identification Of 18 SPL Gene Family Members And Functional Analysis Of BpSPL8 In Betula Platyphylla Suk.

The monoecism and flowering asynchronism of the forest tree Betula platyphylla are important characteristics for research on flower development in forest trees.SQUAMOSAPROMOTER BINDING PROTEIN-LIKE(SPL)proteins are the plant-specific transcription factors and participate in different pathways,including the vegetative to reproductive transition,male sterility,biosynthesis of GA,plant morphogenesis and response to environmental stress.Eighteen full-length open read frames(ORFs)of SPL genes in Betula platyphylla were identified from the transcriptome of normal male inflorescences(NM),normal female inflorescences(NF)and mutant male inflorescences(MM),and named successively BpSPL1 to BpSPL18.Phylogenetic analysis of the BpSPLs,AtSPLs and PtSPLs divided them into eight groups,reflecting that the evolutionary relationship between woody plants is closer than that between woody plant and herbaceous plant.Motif analysis of the full-length amino acid sequence of SPLs in Betula platyphylla indicated that other motifs besides the SBP domain are also related to the function of the SPL genes.Real-time fluorescent quantitative PCR(qRTPCR)analysis of BpSPL genes in different tissues showed that most are similar in structure with a similar expression pattern,and others that have a close evolutionary relationship differ in expression pattern.These results indicated that the gene structure determines the intercommunity and specificity of the function.The online tool psRNATarget using the BpmiR156 sequence predicted that miR156 targeted BpSPL genes,and we found 10 BpSPL genes that were mediated by miR156.The miR156 binding site of BpSPL2,BpSPL3,BpSPL4,BpSPL6,BpSPL9,BpSPL10 and BpSPL12 is in the coding region of each,and that of BpSPL1,BpSPL13 and BpSPL15 is in the 3? UTR.These results provide the molecular basis for further functional study of SPL genes in Betula platyphylla and is also a supplement to the systematic analysis of SPL genes in forest tree species.Bioinformatics analysis of BpSPL8 indicated that BpSPL8 is a intracellular and hydrophobic protein which located in nucleus.There is no random coil structure and it may contain two transmembrane domains,that one model scroed higer with 19 amino acid residues and the N-terminal outside the membrane.The other with 19 amino acid residues also and the N-terminal within the membrane.The BpSPL8 protein may cotain 2 N-glycosylation sites and 11 O-glycosylation sites.We constructed an overexpressed BpSPL8 transgenic Arabidopsis that fusion expressed with GFP mediated by Agrobacterium,and confirmed that BpSPL8 is a transcription factor with the transcriptional activation activity and locates in the nucleus.Functional analysis of BpSPL8 displayed that it involves in regulating different development progresses:(1),BpSPL8 can delay flowering by reducing the sensitivity to GA under short day condition;(2),BpSPL8 controls the number and morphogenesis of leaves,including up-rolling leaves under long day condition and folded leaves mediated by GA under short day;(3),Bp SPL8 can promote the root elongation in the late development of root and also inhibit lateral root formation;(4),BpSPL8 may involved in regulating the biosynthesis of carotenoid and secretion metabolism.These results show that there is a complex regulation network in the SPL family genes mediated by other components which may provide a new idea on the functional research of SPL genes.