Cloning And Functional Analysis Of Flowering Related Genes PheEMF2,PheFT And PheTFL1 From Moso Bamboo

The fast growth and great regeneration ability make Moso bamboo(Phyllostachys edulis)become the important shoot and timber bamboo specie in our country,which has enormous economic,social and ecological values.The bloom cycles of the bamboo are long,and the flowering time and space are unpredictable,while it will die after flowering,so it's difficult to carry out genetic variation and breeding improvement through conventional methods.The structure,function,and expression patterns of the genes related to moso bamboo flowering were studied to find their functions in the flowering pathway,which was expected to used for shortening the flowering cycle through genetic engineering.This research not only could eliminated the cross-breeding barriers created by the long flowering cycle but also could provide theoretical basis for further study on the flowering mechanism of moso bamboo.PheEMF2 and PheTFL1 genes were cloned from the young panicle of moso bamboo through RT-PCR.The ORF of PheEMF2 contained 1821 nucleotides coding a putative protein composed of 606 amino acids.This putative protein belonged to EMF2 subfamily in PcG protein family,which included conserved C2H2 zinc-finger and VEF-box domain.The prokaryotic expression vector pET30a-PheEMF2 was constructed to transfer into Rosetta(E.coli),and the fusion protein was about 72 kD by IPTG inducement.The plant over-expression vector pCAMBIA2300-PheEMF2 was constructed to transfer into wild-type arabidopsis,and two transgenic lines PEMF2-1/2 were screened successfully which had the same phenotype of delaying flowering.The ORF of PheTFL1 gene contained 522 nucleotides encoding a putative protein composed of 173 amino acid.This putative protein belonged to TFL1 subfamily in PEBP protein family,which includeed conserved D-P-D-X-P and G-X-H-R motifs,His88 and Asp 144 residues and END triplet motif.The expression vector of Sub-cellular localization 35S::PheTFL1:YFP was constructed to carry out transient expression in arabidopsis protoplast.The results tested by laser scanning confocal microscope show that the fluorescent signals of PheTFLl::YFP fusion proteins are distributed in the cytoplasm and cell membrane.The plant over-expression vector pCAMBIA2300-PheTFL1 was constructed to transfer into wild-type arabidopsis,but the transgenic lines have no significant phynotype compared with wild-type arabidopsis.The prokaryotic expression vector pET30a-PheTFL1 was constructed,which is induced to obtain about 72 kD of fusion protein.The fusion proteins were partially soluble,but most of them were in form of inclusion body in the cytoplasm.The purified fusion protein from prokaryotic expression though Ni-chelating affinity chromatography method immunized the rabbits and antibody titer was tested by enzyme-linked immunoabsorbent assay(ELISA)method.The results indicate antibody titer of PheTFL1 is 1:51200.PheFT gene was cloned from the young panicle of moso bamboo through rapid-amplification PCR of cDNA ends.PheFT gene contained 540 nucleotides encoding 179 amino acid.This putative protein belonged to TFL1 subfamily in PEBP protein family,which includeed conserved D-P-D-X-P and G-X-H-R motifs,Tyr85 and Glnl40 residues and highly conserved LYN triplet motif.The expression vector of Sub-cellular localization 35S::PheFT:YFP was constructed,and the results of transient expression in arabidopsis protoplast show that the fluorescent signals of PheTFL1::YFP fusion proteins are distributed in the cytoplasm,cell membrane and chloroplast.The prokaryotic expression vector pET30a-PheFT was constructed,which is induced to obtain about 26 kD of fusion proteins,and most of them were insoluble.The purified fusion protein though conventional inclusion body purification method immunized the rabbits and its antibody titer is same with PheTFL1 antibody.The house-keeping genes of ACT2?EFl??GAPDA?TUA?TUB?UBC18?PP2A and UBQ in arabidopsis have the relative stable expression level.According to their sequences,the sequences of homologous genes in moso bamboo were found in genbank as candidate genes of this study.The primers used in qRT-PCR were designed on the basis of their conserved sequences.CT value from qRT-PCR was used to review the relative stable expression level of nine candidate genes in different tissues and organs of seedling,non-flowering bamboo and flowering bamboo.According to composite analysis of the results of geNorm and NormFinder programs,PP2A was selected for analyzing the relative expression level of target genes,because of its stable expression level in different tissues and organs.lng RNA extracted from different tissues and organs of seedling,non-flowering bamboo and flowering bamboo was used to the synthesis of cDNA first chain.2?l cDNA samples was used to analyze the relative expression level of target genes.The results revealed that PheEMF2 gene had high expression level in young immature floret,and PheTFL1 expressed at high level in young immature floret and tender organs.PheFT had high expression level in leaves and branches of flowering bamboo.PheTFL1 and PheFT were homologous genes,but they had different expression level in the tissues and organs.