Cloning And Analysis Of 14-3-3 Protein Genes In Haloxylon Ammodendron

14-3-3 proteins are an important regulatory proteins within the organism, which have an important regulatory role on cell metabolism and circulation, signal transduction, transcriptional regulation, protein transmembrane transport, as well as a variety of physiological processes biotic and abiotic stresses. This paper cloned the four 14-3-3 protein genes by RT-PCR method from Haloxylon ammodendron,and then analyzed their preliminary bioinformatics. This paper explored the gene expression characteristics in different tissues of Haloxylon ammodendron and under drought stress by qRT-PCR method and then analyzed the subcellular localization by PEG mediated transient expression in Chickpeas protoplasts. The results showed as follows:(1) The four 14-3-3 protein gene were cloned from Haloxylon ammodendron, named HaFT-1, HaFT-2, HaFT-3 and HaFT-4. The ORF of the 14-3-3 protein genes were respectively 795 bp, 792 bp, 792 bp, 786 bp, respectively encoded a protein of 264, 263, 263, 261 amino acids, and then respectively analyzed and forecasted their bioinformatics. The results showed that these 4 proteins were hydrophilic and unstable protein with similar physical and chemical properties, but without transmembrane structure. The homology of amino acid sequence revealed that the homology of the four 14-3-3 proteins were the highest with Sugar beet above 85%. Molecular phylogenetic tree showed that HaFT-1, HaFT-3 with Sugar beet and Gossypium hirsutum clustered into one category; HaFT-2 with Sugar beet, Mesembryanthemum crystallinum clustered into one category; HaFT-4 with Sugar beet gather alone together.(2) Promoter sequence(1793 bp) of HaFT-3 gene was cloned from Haloxylon ammodendron by genome walking method. Sequence analysis showed that the promoter included cis-acting elements about plant hormones, drought, light, heat and other various external stimuli, as well as the elements involved in plant growth and development.(3) The expression of four 14-3-3 genes in different tissues(root, assimilating shoots, flowers and seeds) of Haloxylon ammodendron were analyzed using 18 S rRNA as reference gene. The results showed that HaFT-1 expression was highest in the roots and seeds followed by. The expression of HaFT-2 and HaFT-4 were the highest in assimilating shoots, the amount of expression of the root followed. The expression of HaFT-3 in roots, assimilation branches, seeds and flowers was decreasing in turn.The espression of HaFT-1, HaFT-2, HaFT-3 and HaFT-4 under the stress of drought by qRT-PCR. The expression of HaFT-1, HaFT-2, HaFT-3 and HaFT-4 were respectively the highest with the soil water content at 9 %, 12 %, 3 %. But the expression of HaFT-2 levels was relatively lower. The expression of HaFT-3 and HaFT-4 showed an increasing trend.(4) Plant expression vector was successfully constructed and respectively named p CAMBIA1304-HaFT-1, pCAMBIA1304-HaFT-2, pCAMBIA1304-HaFT-3 and pCAMBI A1304-Ha FT-4. The expression vector was successfully transferred into Chickpeas prot oplasts to express the transient expression. The results showed that HaFT-1, HaFT-2, HaFT-3 and HaFT-4 had the nuclear localization function.