Cloning And Expression Analysis Of Late Bolting BcFLC3and BcFLC3Genes From Non-Heading Chinese Cabbage

The PCR primers were designed on the convervation domain encoding known FLC gene,a set of FLC genes were obtained from Brassica campestris ssp. chinensis by the homology-based cloning technique.In order to research these genes better, to regulate and control bolting and flowering. In this study, we use real-time PCR to finish the expression analysis at different developmental stages and different parts of the plant,use Southern hybridization technique to research the copies of BcFLC3gene in Brassica campestris ssp. chinensis’s genome.It provides theoretical and technical support for further using the gene to regulate vegetable’s late bolting.1. cloning and characterization of BcFLCl gene from Brassica campestris ssp. chinensisMaterial ’NJ074’ as experimental material is provided by Cabbage Research Group of College of Horticulture, Nanjing Agricultural University.Referencing to Brassica napus FLC1gene (GenBank accession number AY036888.1) designed specific primers.BcFLC1genes were obtained from late bolting Brassica campestris ssp. chinensis ’NJ074’ by RT-PCR,5’RACE,3’RACE technique. The full-length cDNA sequence of909bp in length contained an open reading frame of576bp.Using bioinformatics methods and variety of online software to analyse BcFLC1’s protein amino acids、bolting related gene’s homology with other plants、 phylogeny、 hydrophilic, transmembrane domains、 subcellular localization, and protein structure. Using DNAMAN software tp analyse BcFLC1gene encoding the amino acid sequence with bolting related gene homology of other plants. The results show that, there are varying degrees of homology between BcFLC1and Bolting-related genes of other plants., further comparison to genes Brassica napus、 Brassica rapa subsp. Pekinensis、 Raphanus sativus、 Aabidopsis thaliana showed the identity were96%、95%、82%、86%. Analysis of physical and chemical properties show that it encoded194amino acids with PI of5.61, a molecular weight of21877.6D. TMpred transmembrane analysis shows that, It contains a remarkable transmembrane helix area, This region and the predicted hydrophobic Protscale have basic overlap. Target P server and WOLFPSORT server program predicted that the protein was cytoplasmic protein. Domain analysis showed that the protein contained multiple phosphorylation sites. Secondary structure is a-helix and random coil mainly.To predicted the domain of BcFLCl protein, The results showed that the gene is MADS-box genes, which encoded proteins that1～60aa belong to MADS box protein. The study can provide a theoretical basis for experimental research on structure and function of BcFLC1proteins.Real-time PCR analysis showed expression level of the BcFLC1gene in unbolting plant’s leaves was higher than that of bolting plant. The expression level of different tissues in bolting plant existed differences, The expression level from high to low are leaf, stem, bud, flower and root.2. cloning and characterization of BcFLC3gene from Brassica campestris ssp. chinensisReferencing to Brassica rapa subsp. Pekinensis FLC3gene (GenBank accession number AY036890.1) designed specific primers.BcFLCl genes were obtained from late bolting Brassica campestris ssp. chinensis ’NJ074’ by RT-PCR,5’RACE,3’RACE technique. Using DNAMAN software to analyse the sequence, The full-length cDNA sequence of1017bp in length contained an open reading frame of576bp.Its ORF (open read frame) encoded a polypeptide of197amino acids.Using bioinformatics methods and variety of online software to analyse BcFLC3’s protein amino acids、bolting related gene’s homology with other plants、 phylogeny、 hydrophilic, transmembrane domains、 subcellular localization, and protein structure. Using DNAMAN software tp analyse BcFLC3gene encoding the amino acid sequence with bolting related gene homology of other plants. The results show that,there are varying degrees of homology between BcFLC3and Bolting-related genes of other plants., further comparison to genes Brassica oleracea var. capitata、Raphanus sativus showed the identity were94%and79%.Analysis of physical and chemical properties show that it encoded197amino acids with PI of9.36, a molecular weight of21.62KD.TMpred transmembrane analysis shows that, It contains a remarkable transmembrane helix area, This region and the predicted hydrophobic ProtSCale have basic overlap. Target P server and WOLFPSORT server program predicted that the protein was cytoplasmic protein. Domain analysis showed that the protein contained multiple phosphorylation sites. Secondary structure is a-helix and random coil mainly.To predicted the domain of BcFLC3protein, The results showed that the gene is MADS-box genes, which encoded proteins that1～60aa belong to MADS box protein. The study can provide a theoretical basis for experimental research on structure and function of BcFLC3proteins.Real-time PCR analysis showed expression level of the BcFLC1gene in unbolting plant’s leaves was higher than that of bolting plant. The expression level of different tissues in bolting plant existed differences, The expression level from high to low are leaf, stem, bud, flower and root.Southern blot showed that BcFLC3of Brassica campestris ssp. chinensis genome is multicopy.