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

LI Xiang;HOU Lu;KANG Yaxuan;PANG Xiaoming;LI Yingyue;College of Biological Sciences and Biotechnology, Beijing Forestry University·National Engineering Laboratory for Tree Breeding·Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plant, Ministry of Education;

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 c DNA was prepared from 2 μg total RNA using the First Strand c DNA Synthesis Kit and then used to amplify FT sequence. It was conducted in a total volume of 20 μL containing 1 μL c DNA(around 1 μg), 4 μL forward primer(10 μmol·L-1), 4μL reverse primer(10 μmol·L-1), 10 μL 2×PCR Mix and 1 μL dd H2 O. The reaction was programmed in a thermal cycler and conducted under condition of initial 5 min denaturation at 94 ℃, 35 cycles of 94 ℃ for30 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 gelMaxi Purification kit. The PCR purified products and p BI-121 vector were digested by Xba I, Sma I be-fore being mixed with T4 DNA ligase and adaptor at 37 ℃ continue for 30 min. Then they were transferredinto E. coli strains DH5α competent cells, and finally plated onto LB(Luria-Bertani) agar containing 100μg·m L-1 ampicillin, 0.5 mmol·L-1 IPTG(Isopropyl β-D-1-thiogalactopyranoside) and 80 μg·m L-1 X-Gal. Plates were incubated at 37 ℃ overnight. The positive clones were selected for PCR identifying andsequencing by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. And then, weperformed bioinformatics analysis of the gene. The protein sequences of the gene were predicted with theNCBI ORF(open reading frame) program. PI(theoretical isoelectric point) and Mw(molecular weight) ofthe protein were predicted using online Ex PASy proteomics tools.org/protparam/). The tertiary structure ofthe FT proteins was predicted using Phyre2(http://www.sbg.bio. ic.ac.uk/phyre2) software, and viewedwith Ras Mol 2.7.2.1. The BLAST algorithm was used to search the NCBI Gen Bank(http://www.ncbi.nlm.nih.gov/) databases for homologous sequences and ascertain the identity of target gene. The amino acid se-quences of FT homologue gene were analyzed using the Clustal X multiple sequence alignment programver. 1.83 and Bio Edit ver.7.7.0(http://www.mbio.Ncs u.edu/Bio Edit/bioedit. html). The NJ(Neighbor Join-ing) tree was constructed by MEGA 6 software. Quantitative real-time PCR was performed usingSYBR?Premix Ex Taq? 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) andplate reading after each cycle. Data were analyzed using ABI 7500 Real-time PCR system Gene Expres-sion software and presented as a mean ± SD. RT-PCR products were detected by 1.5% agarose gel andchecked with the fluorescence intensity of a certain temperature period. Relative expressions of all repli-cas of each sample were calculated using the 2-△△Ctmethod.【Results】A FT homologous gene was isolatedfrom Chinese jujube and designated as Zj FT. The relative expression was analyzed in different tissues andorgans. The sequence structure analysis revealed that Zj FT 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 andMw was 9.68 and 18.112 ku, respectively, which indicated Zj FT 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 inamino acid sequences, respectively. Meanwhile, the Phylogenetic analysis illustrated that Zj FT was most-ly closed to Md FT in Malus domestica. The secondary structure of Zj FT amino acids possessed Alpha-he-lical(32.7%), extension chains(23.9%), β-rotation(8.18%) and irregular curls(8.18%). The main compo-nent of the secondary structure of Zj FT protein was irregular curls. 3 D structure included 2 alpha helicesand 5 β-folding areas. There was a PEBP gene super family in Zj FT structural domain. The structure of‘Dongzao'jujube FT protein was similar to that of Malus domestica FT protein. Real-time quantitativePCR indicated that the Zj FT were expressed in different growth stages of nutritive organs and reproduc-tive organs. The relative expression of nutritive organs was lower than that in reproductive organs.【Conclu-sion】The FT gene was successfully cloned in‘Dongzao'jujube and its accession number of Gen Bank isKR872844. The results suggested that FT gene played a momentous role in vegetative growth and repro-ductive development. Compared with other flowering gene, Zj FT was considered to be conservative and itcould regulate the process of flower development. The relative expression of Zj FT gene in the reproductiveorgans was higher than that in vegetative organs. The highest expression level was found in Fruits. The ex-pression of Zj FT gene showed a monthly decline trend in leaves. It would be worthwhile to transfer the Zj FT gene into Arabidopsis to further explore its function.