Cloning And Characterization Of Mother Of FT (MFT) Gene In Peanut (Arachis Hypogaea L.)

MOTHER OF FT（MFT）of Arachis hypogaea L. encodes a phosphatidylethanolamine–binding protein PEBP, and has a conserved domain, belonging to thePEBP gene family. Chardon et al. systematically analyzed the PEBP gene familay inplant, and found that this family includes three subfamilies: FT-like, TFL-like andMFT-like. FT-like subfamily has two members: FT (FLOWERING LOCUS T) andTSF (TWIN SISTER OF FT). TFL-like subfamily has three members: TFLl(TERMINAL FLOWERl)、ATC (ARABIDOPSIS CENTRORADIALIS HOMOLOGUE)and BFT (BROTHER OF FT ANDTFL1). MFT-like subfamily has only one member:MFT (MOTHER OF FT AND TFLl). In the FT-like subfamily, FT induces plants toblossom, and TFL inhibits the transformation from vegetative growth to reproductivegrowth and delays plants blossom. The FT/TFL1ratio controls the balance ofindeterminate and determinate growth. MFT has similar function with FT for showingredundancy flower induction effect, and promotes plants flowering and seedgermination. Because of the little known about the MFT, we will take advantages ofthe study on FT to make further research on MFT.We obtained two EST sequence of peanut from the GenBank (Genbank No.GO339983and EG030494), and the two ESTs both have complete open readingframes (ORF). The analyses results showed that the two ESTs have high homologywith PEBP protein, and belong to the MFT-like gene subfamily. We took the twoESTs as templates to design primers and used the young peanut seeds cDNA astemplate for gene cloning. We got two PEBP genes and named as AhMFT1andAhMFT2respectively.Sequence analysis shows that both of AhMFT1and AhMFT2contain a singleORF of522bp. There are17bases differences between the ORFs of the two genes.AhMFT1encodes a protein of173amino acids with an estimated molecular mass of18.883kD and an isoelectric of9.06, while AhMFT2encodes a protein of173amino acids with an estimated molecular mass of18.828kD and an isoelectric of9.09. Sixamino acids differences were detected between the two AhMFT proteins. Theprediction analysis indicate that both of the two AhMFT proteins have a conservedPEBP protein domain located at25-163amino acid, but have no signal peptide andtransmembrane structure, and localize in the cytoplasm.Moreover, we designed primers to clone the full length genome sequences ofAhMFT1and AhMFT2. Results showed that AhMFT1and AhMFT2genomesequences have1450and1487bp respectively,4exons and3introns as well.The expression patterns of AhMFT1and AhMFT2in different organs of peanutwere analyzed by semiquantitative RT-PCR. It was found that the expression level ofAhMFT1and AhMFT2were high in seeds and flowers. According to above results,we presume that AhMFT1and AhMFT2proteins would have more functions inregulation of peanut seeds germination than in promotion of peanut flowering.To investigate the functions of the two AhMFTs in peanut ‘Luhua14’, weconstructed two constitutive overexpression vectors pROKII-35S-AhMFT1andpROKII-35S-AhMFT2to transform the wild-type (WT) tobacco byAgrobacterium-mediated transformation. Twenty-one transgenic tobacco lines wereobtained for AhMFT1, while18lines of transgenic tobacco lines were obtained forAhMFT2.The promoters of AhMFT1and AhMFT2genes were cloned usingGenomeWalker Universal Kit and transgenetic vectors were successfully constructedto test the functions of promoters. To detect the functional elements in the promotersof AhMFT1and AhMFT2genes, we also constructed the vectors losing part domainsfrom the whole promoter.We constructed two kinds of the subcellular localization vectorspBSK-35S-AhMFT-EGFP and pBSK-35S-AhMFT-RFP to test the subcellularlocalization of the two AhMFTs. By the biolistic bombardment experiments, theAhMFTs were instantly expressed in the onion epidermal cells and were observedwith the Laser Scanning Confocal Microscope. The results indicated that AhMFT1and AhMFT2are located in the whole cells with the strongest signal in the nucleus. The results are similar to the control. We used RFP as report gene to confirm the GFPexperiment results, and we obtained the same results.