Cloning,expression And Functional Analysis Of Flowering Related SPL Gene In Betula Luminifera

As a specific transcription factor family in plant, SBP-box plays critical roles in regulation of floral development. In this study, thirteen SPL genes were isolated from a precious timber tree Betula luminifera through RACE strategy. Through bioformatic analysis, subcellular location, expression pattern analysis and Yeast two hybrid, the functions of these 13 BlSPLs were investigated, and the major conclusion are listed as following.1. Sequence analysis revealed that the length of Bl SPL cDNAs varied between 830(Bl SPL3) and 4,234 bp(BlSPL5). Among these genes, BlSPL5 has largest open reading frame(3249 bp), encoding 1082 amino acids. The phylogenetic analysis showed these BlSPLs genes belonged to 8 different clades of SPL family, among that BlSPL4, BlSPL5 and BlSPL12, Bl SPL6 and Bl SPL7, Bl SPL1 and BlSPL9, BlSPL11 and Bl SPL13 belonged to the same clade, respectively.2. Using RLM-RACE method, the miR156/157 cleavage sites of BlSPL1, Bl SPL3, BlSPL6, BlSPL7, BlSPL8, BlSPL9 and Bl SPL11 were verified. The results show that, in addition to the cleavage site of BlSPL8 miR156/157 is at 11/12 th base pair, the other BlSPL’s cleavage site were all at 10/11 th base pair; and in the cleavage sites, except BlSPL9, all the genes have a mismatched base to miR156.3. Subcellular location analysis was also performed for several BlSPLs genes. The result indicated that the miR156 targeted Bl SPL1, 3 and 11 were located at nuclear, while the mi R156 nontargeted BlSPL5 was mainly detected in cytoplasm.4. Expression patterns of SPL genes and miR156 in different tissues, organs, different age plants and different developmental stages of inflorescence were investigated using Real time-PCR. The result showed that the expression patterns of BlSPL gene are obviously different. Especially, the expressions of mi R156 and miR156 targeted Bl SPLs in different age plants showed apparent negative relationship. This means that miR156 and BlSPLs may also play important role during juvenile phase transition in Betula luminifera.5. Through Yeast Two-Hybrid, the interaction between BlSPL and Bl DELLA were determined. The result showed that Bl SPL1、5、13 could interacted with DELLA. This suggested the these complex may involved in the GA mediated floral pathway.