Gene cloning and functional identification of AfFT3 gene in Aechmea fasciata(Lindl.)Baker
[Objective]The flowering-related gene AfFT3 in Aechmea fasciata(Lindl.)Baker was cloned and its bio-logical function was identified,which provided a theoretical basis for further analysis of the molecular mechanism of flowering of Bromeliaceae plants.[Method]AfFT3 gene was cloned and its sequence was analyzed by bioinformatics soft-ware.The expression levels of AfFT3 at low temperature(18℃),normal temperature(25℃)and high temperature(35℃)and exogenous ethylene treatment were detected by real-time fluorescence quantitative PCR(qRT-PCR).AfFT3 gene was transferred into Arabidopsis thaliana(L.)Heynh by Agrobacterium flower dipping.The phenotype of transgenic plants was observed,and its biological function was predicted by heterologous expression.The interaction between the promoter and AfEIN3 protein was preliminarily identified by yeast one-hybrid assay.[Result]The full length of AfFT3 gene cloned from A.fasciata was 570 bp,and the coding sequence(CDS)was 465 bp,encoding 154 amino acid resi-dues.AfFT3 protein had a molecular weight of 17.3 kD,was a stable hydrophilic protein without a transmembrane helical region and contained several phosphorylation sites of protein kinases including PKC,PKA,cdc2,INSR and GSK3.The homologous sequence alignment revealed that two amino acid residues of AfFT3 protein 138Trp and 140Gln mutated to 138Met and 140Glu.Phylogenetic analysis indicated that AfFT3 protein belonged to the FT-like protein subfamily of the PEBP family,and was close to the TFL-like subfamily.Compared with the control(plants cultivated in normal conditions in greenhouse),the relative expression of AfFT3 gene in A.fasciata was increased significantly(P<0.05,the same below)or extremely significantly(P<0.01,the same below)under the treatment of high temperature and low temperature.Under the conditions of high temperature,normal temperature and low temperature,after exogenous ethylene treatment,the rela-tive expression of AfFT3 gene in A.fasciata was increased significantly or extremely significantly compared with the con-trol,with the highest relative expression at normal temperature.A total of six transgenic A.thaliana plants(L1 to L6)were obtained,in which the transgenic plants L3 and L6 were about 8 d delayed than the empty vector and wild-type A.thaliana.The AfFT3 gene promoter sequences(AfFT3-P1,AfFT3-P2 and AfFT3-P3)were obtained by segmented amplification with the lengths of 390,1077 and 552 bp respectively.The results of yeast one-hybrid experiment preliminarily suggested that promoter sequence AfFT3-P1 and AfFT3-P3 might interact with AfEIN3 protein.[Conclusion]The effective expres-sion of AfFT3 gene is induced by high temperature,low temperature stress and ethylene in different degrees,but the in-duction effect of ethylene on the expression of AfFT3 gene under high temperature and low temperature conditions is lower than that of normal temperature.The genetically modified plants delay bolting,indicating that AfFT3 gene is in-volved in the regulation of flowering process of A.fasciata,and respond to temperature and ethylene signals.
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