Cloning And Analysing Of 14-3-3 Protein HaFT-5?9 Genes And Functional Verification Of HaFT-4 Gene In Haloxylon Ammodendron

14-3-3 protein are a kind protein family, which is highly conserved, multigene encoded, presented as homologous or heterologous dimers and has a regulatory function. 14-3-3 protein plays an important role in the nuclear localization of transcription factors, regulating the expression of genes, enzyme activity regulation and a variety of cellular activities. In this research, five 14-3-3 protein genes were successfully cloned by using RT-PCR method from Haloxylon and preliminary analyzed with Bioinformatics method.We studied expression levels of the five genes under drought stress by Fluorescent quantitative q RT-PCR,obtained and analyzed the Ha FT-4 transgenic tobacco plants by Agrobacterium mediated transformation.The results are as follows:1. Five Haloxylon 14-3-3 protein genes were successfully cloned, named as Ha FT-5, Ha FT-6, Ha FT-7,Ha FT-8 and Ha FT-9. The ORF length of five sequences were 420 bp, 681 bp, 789 bp, 792 bp and 789 bp and respectively encoded 139, 226, 262, 263 and 262 amino acids. The five amino acid sequences were analyzed by Bioinformatics method. The results showed that Ha FT-5 was a stable protein and the other four proteins were unstable. All five proteins were hydrophilic, did not exist signal peptide and transmembrane. After sequence similarity alignments, we found that Ha FT-5, Ha FT-6 had the highest homology with beet and Ha FT-7,Ha FT-8,Ha FT-9 had the highest homology with spinach. Phylogenetic tree showed that Ha FT-5 and beet were clustered into one, Ha FT-6 with grapes and lily were clustered into one, Ha FT-7, Ha FT-8,Ha FT-9 with spinach were clustered into one.2. The expression levels of five 14-3-3 protein genes were analyzed by using real-time fluorescence quantitative under 20% PEG6000 stress for 0 h, 4 h, 8 h, 12 h and 24 h. Results showed that the expression of Ha FT-5 gene came to the highest at 12 h and then decreased significantly. Compared with the expression level of unstressed Haloxylon, expression levels of Ha FT-9 gene had the most significant change, but expression levels of Ha FT-6 gene did not change significantly.3. Five transgenic tobacco plants were obtained by successfully constructing a p CAMBIA1304-Ha FT-4 plant expression vector and transferring it into tobacco with Agrobacterium mediated transformation method. The changes of MDA, proline, soluble sugar and chlorophyll contents were analyzed under drought, salt and cold stress with three transgenic and a wild-type tobacco plant leaves. The results showed that the three Ha FT-4 transgenic plants had a similar trend after stress. But compared to wild-type tobacco plants, their MDA content decreased, proline content increased, Soluble sugar content increased, chlorophyll content increased. On the whole, the Ha FT-4 transgenic tobaccos had a stronger ability to resist stress.