Study For FLC Gene In Poplar——Bioinformatics Analysis Of FLC Gene Family In Populus Trichocarpa And Cloning And Expression Analysis Of PnDLC1

Recently, substantial advance was observed in research of floral induction in model plant Arabidopsis thalina. It contains four main pathways:Photoperiod pathway, vernalization pathway, autonomous pathway and GA pathway. As the study of the floral pathway is clearer, people try to get the relation between Arabidopsis and trees. Recent evidence showing the extent to which LYF and FT is effective in promoting flowering in trees is discussed, and its effectiveness in orange tree and poplar is reported. Extensive research shows that FLC （Flowering Locus C） is a key repressor involved in floral induction pathways in Arabidopsis thaliana. It is also playing an important role in the vernalization pathway and the autonomous pathway. Overexpression of FLC can inhibit the expression of the downstream genes, SOC1and FT, which further inhibits flowering, vice versa. In this paper, results are listed as follows:（1） We characterized and identified13genes of FLC gene family in the poplar genome. Motif-based sequence analysis showed all PtFLCs contain one M domain of MADS-box, some genes have I、K and C domains. It is also shown that PtFLC genes distribute over nine of the19poplar chromosomes, PtrFLC-10（745710） and PtrFLC-11（678188） are located in scaffolds. Silico microarray data analysis suggested that differential expression existed in different tissuses.（2） We identified21early flowering Arabidopsis flc homozygote mutants, the mutants started to flower around7-day earlier compared to wild-type. The mutant （SALK₁03719.45.30.x） is that the AtFLC gene （Genebank Accession:NM₂1052） was knockouted. AtFLC and PtrFLC-13（758707） encoded proteins with similarity of49%. We cloned a PnFLCl gene from Populus simoniixP.nigra according to PtrFLC-13. PnFLCl is726bp, which included three different nucleotides compared to the PtrFLC-13. However, they encoded the same. Quantitative Real-time PCR showed that the gene expression in flower buds gained the highest transcripts, followed by stems and leaves, lowest in the root. After vernalization treatment, the level of gene expression decreased in the roots, stems and leaves.（3） Prokaryotic expression was performed in E.coli for the PnFLCl and the recombinant protein was purified.30℃and8h was the best conditions for protein production. The fusion Protein was Purified used as antigen to immune rabbit for subsequent preparing for polyclonal antibody to PnFLCl. This antibody specifically responsed to PnFLCl.（4） We successfully constructed two kinds of expression vectors, namely overexpressing vector and RNAi vector, which were drived by35S promoter, We also successfully transformed these two vectors to Agrobacterium EHA105, which is important for the genetic transformation in poplar next step.