Research Of G.m14-3-3 Gene In AM Fungi Glomus Mosseae

14-3-3 proteins form a family of highly conserved, acidic, dimeric proteins, these proteins have been identified in all eukaryotic species investigated and involved in almost every cellular process. According to the published EST sequence of 14-3-3 gene from arbuscular mycorrhizal fungal Glomus mosseae, inverse PCR and 3'RACE were used to obtain the 5' and 3'flanking sequence, about 2 kb DNA fragment including the promoter region and 5'UTR was obtained. The Gml4-3-3 gene reported in this study generates two pools of mRNAs that carry a long and a short 3'UTR named G.m14-3-3L and G.m14-3-3S respectively which encode the same G.m14-3-3 protein.The primary and tertiary structure of amino acid sequence deduced from G.m14-3-3 gene were analyzed by bioinformatics. The predicted 3D structure of G.m14-3-3 protein indicates it is similar to 14-3-3 proteins from other species. The putative promoter region of G.m14-3-3 gene was analysised in yeast strain Y187 by constructing a vector carried egfp reporter gene.Real time PCR was used to investigate the transcription pattern of total G.m14-3-3 and G.m14-3-3L, under different environment. The main results are shown as follows:1) The transcription lever of total G.m14-3-3 was little difference between quiescent spore and intraradical mycelium, while the transcription level of G.m14-3-3L was about 0.5-times higher in quiescent spores than that in intraradical mycelium.2) The transcription level of G.m14-3-3 steadily decreased along the 1h,6h,12h,24h after addition of Cu, at the 24 h time-point. About 0.5-times decrease was detected compare with CK, while only a little difference from CK after one week and one month under Cu treatment was observed, G.m14-3-3L and total G.m14-3-3 had the same changing tendency under Cu-stress.3) G.m14-3-3 transcription pattern was a little fluctuation under 60 mg/kg Cd-stress at different time-piont, but after a month under 90 mg/kg Cd treated, the lever of gene transcription was about 0.7-times higher than CK.4) Only slightly decreased transcript levels were measured under supplementation with NaCl at different time and different concentration.That means besides regulating at transcriptional level, post-transcriptional regulation may also play an important role in G.m14-3-3 gene expression.The G.m14-3-3 gene ORF was cloned into pET28a(+) to investigate the expression in E.coli BL21(DE3), a recombinant protein about 30 kDa was induced successfully, the partial fragment on genomic DNA of 14-3-3 gene from other three AM fungi Gigaspora margarita, Glomus intraradices, Gigaspora rosea were obtained by designing primers on highly conserved domains. This research makes a good foundation for subsequent research on signal pathway involved in 14-3-3 protein in AM fungi.