Cloning and expression analysis of FT homologous gene in Ziziphus jujuba Mill.‘ Dongzao’

Ziziphus jujuba Mill.‘Dongzao’%Flower development%FT gene%Cloning%Expression

[Objective] PEBP (phosphatidyl ethanolamine-binding protein) family proteins exist widely in plants and play an important role in controlling flower development.The PEBP family was divided into three major categories:FT(Flowering locus T),TFL1 (TERMINAL FLOWER 1) and MFT(MOTHER OF FT AND THL1).The FT gene is very conservative among different plants,and its homologous gene can shorten the flowering time of plants.To explore the process of flower development of Chinese jujube (Ziziphus jujuba Mill.‘ Dongzao’),the FT gene was cloned in this study,its function and expression pattern in different tissues and developmental stages were analyzed.[Methods] Total RNA was extracted from shoot apices,terminal buds,young leaves,mature leaves,flower buds,flowers and fruits by CTAB (Hexadecyl Trimethyl Ammonium Bromide)and Kit.First-strand cDNA was prepared from 2 μg total RNA using the First Strand cDNA Synthesis Kit and then used to amplify FT sequence.It was conducted in a total volume of 20 μL containing 1 μL cDNA (around 1 μg),4 μL forward primer (10 μmol· L-1),4 μL reverse primer (10 μmol· L-1),10 μL 2×PCR Mix and 1 μL ddH2O.The reaction was programmed in a thermal cycler and conducted under condition of initial 5 min denaturation at 94 ℃,35 cycles of 94 ℃ for 30 s,54 ℃ for 30 s,72 ℃ for 30 s and extension for 10 min at 72 ℃.PCR products were fractionated using 1.0% agarose gel and photographed.Amplified fragments were purified and retrieved by the TIAN gel Maxi Purification kit.The PCR purified products and pBI-121 vector were digested by Xba Ⅰ,Sma Ⅰ before being mixed with T4 DNA ligase and adaptor at 37 ℃ continue for 30 min.Then they were transferred into E.coli strains DHSα competent cells,and finally plated onto LB (Luria-Bertani) agar containing 100 μg· mL-1 ampicillin,0.5 mmol· L-1 IPTG (Isopropyl β-D-1-thiogalactopyranoside) and 80 μg· mL-1 X-Gal.Plates were incubated at 37 ℃ overnight.The positive clones were selected for PCR identifying and sequencing by Shanghai Sangon Biological Engineering Technology and Services Co.Ltd.And then,we performed bioinformatics analysis of the gene.The protein sequences of the gene were predicted with the NCBI ORF (open reading frame) program.PI (theoretical isoelectric point) and Mw (molecular weight) of the protein were predicted using online ExPASy proteomics tools.org/protparam/).The tertiary structure of the FT proteins was predicted using Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2) software,and viewed with RasMol 2.7.2.1.The BLAST algorithm was used to search the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) databases for homologous sequences and ascertain the identity of target gene.The amino acid sequences of FT homologue gene were analyzed using the Clustal X multiple sequence alignment program ver.1.83 and Bio Edit ver.7.7.0 (http://www.mbio.Ncs u.edu/BioEdit/bioedit.html).The NJ (Neighbor Joining) tree was constructed by MEGA 6 software.Quantitative real-time PCR was performed using SYBR(R)Premix Ex TaqTM Ⅱ and ABI 7500 fluorogenic Quantitative PCR.Amplification conditions were:96 ℃ for 1 min,followed by 40 cycles of amplification (95 ℃ for 15 s,60 ℃ for 15 s,72 ℃ for 45 s) and plate reading after each cycle.Data were analyzed using ABI 7500 Real-time PCR system Gene Expression software and presented as a mean ± SD.RT-PCR products were detected by 1.5% agarose gel and checked with the fluorescence intensity of a certain temperature period.Relative expressions of all replicas of each sample were calculated using the 2-△△Ct method.[Results] AFT homologous gene was isolated from Chinese jujube and designated as ZjFT.The relative expression was analyzed in different tissues and organs.The sequence structure analysis revealed that ZjFT gene contained 57 bp 3’-UTR,136 bp 5’-UTR and 525 bp open reading frame.It encoded 174 protein of amino acids.The theory value of PI and Mw was 9.68 and 18.112 ku,respectively,which indicated ZjFT edited a basic and hydrophilic protein.The sequence alignment showed that the similarity between FT gene and Malus domestica,Populus nigra,Litchi chinensis and Vitis vinifera homologues exhibited 98.6%,97.9%,97.2% and 96.18% identities in amino acid sequences,respectively.Meanwhile,the Phylogenetic analysis illustrated that ZjFT was mostly closed to MdFT in Malus domestica.The secondary structure of ZjFT amino acids possessed Alpha-helical (32.7%),extension chains (23.9%),β-rotation (8.18%) and irregular curls (8.18%).The main component of the secondary structure of ZjFT protein was irregular curls.3D structure included 2 alpha helices and 5 β-folding areas.There was a PEBP gene super family in ZjFT structural domain.The structure of ‘Dongzao’ jujube FT protein was similar to that of Malus domestica FT protein.Real-time quantitative PCR indicated that the ZjFT were expressed in different growth stages of nutritive organs and reproductive organs.The relative expression of nutritive organs was lower than that in reproductive organs.[Conclusion] The FT gene was successfully cloned in ‘Dongzao’ jujube and its accession number of GenBank is KR872844.The results suggested that FT gene played a momentous role in vegetative growth and reproductive development.Compared with other flowering gene,ZjFT was considered to be conservative and it could regulate the process of flower development.The relative expression of ZjFT gene in the reproductive organs was higher than that in vegetative organs.The highest expression level was found in Fruits.The expression of ZjFT gene showed a monthly decline trend in leaves.It would be worthwhile to transfer the ZjFT gene into Arabidopsis to further explore its function.