Functional Analysis Of An Interactional Protein GmTPR1 With Stress-Related Transcription Factor GmDREB5 And Isolation Of Stress Responsive Genes Using Nuclear Transportation Trap

Ⅰ. Functional analysis of interactional protein GmTPR1 with stress-related transcription factor GmDREB5Transcription factors play important role in the regulation of plant growth, development, and response to environment. The DREB (dehydration resistance element binding protein) is a family of stress-related transcription factors. DREB family can enhance plant tolerance to abiotic stresses. The activity of DREB is regulated by some specific interactive proteins. Therefore, isolation of genes coding DREB interactive proteins will be contributed for the clarifying of the regulative mechanisms of DREB transcription factors on translational level. In previous research, overexpression of GmDREB5 was proved to enhance stress tolerances of transgenic plants. In order to study regulative mechism of GmDREB5, GmTPR1 gene coding GmDREB5 interactive protein had been isolated by screening cDNA library of soybean treated with drought using yeast-two-hybrid systemt. So far, few members of TPR gene family were studied and functions of most members are unknown. In this study, characteristic and function of GmTPR1 were analyzed. Some main results have been achieved as follows:1. Isolation and phylogenetic analysis of GmTPR1 gene A gene coding GmDREB5 interactive protein, GmTPR1, was isolated from soybean cDNA library. The GmTPR1 protein has one TPR (tetratricopeptide repeat) motif. The homologic analysis of the TPR motif with the similar Arabidopsis indicated that the highest identity of with other TPR proteins was 14% only, suggesting that GmTPR1 is a novel member of TPR protein family.2. Identification of the interaction between GmTPR1 and GmDREB5 The fragment encoding GmTPR1 was inserted into pEASY-E1 (with 6×His in N'end) and the fragment encoding GmDREB5 was inserted to pGEX 4T-1 (with GST tag), respectively to form fusion expression vector,which was analyzed by pull-down and western blot .and results suggested GmTPR1 interacts with GmDREB5 obviously in vitro; full-length sequences of GmTPR1 and GmDREB5 were cloned into the pUCSPYNE and pUCSPYCE BIFC (Bimolecular fluorescence complementation) vectors, respectively,for transient expression analysis in onion (Allium cepa) epidermal cells using the particle bombardment,and fluorescence was observed with a laser scanning confocal microscope. Result indicated that GmTPR1 interacts with GmDREB5 in vivo strongly also.3. Subcellular localization assay of the GmTPR1 and GmDREB5 GmTPR1 and GmDREB5 under the control of the cauliflower mosaic virus (CaMV) 35S promoter were inserted to 163hGFP vector before the 5'end of the coding region of green fluorescence protein (GFP) gene. The fused of plasmids 163hGFP- GmTPR1 and 163hGFP-GmDREB5 were transformated into onion epidermal cells for transient activity analysis. The results indicated that GmTPR1 is located in nuclei and GmDREB5 in the nuclei and membrane, suggesting this two proteins might interact each other in the nuclei of plant.4. Expression pattern analysis of GmTPR1 RT-PCR and Real-time PCR results showed that the expression of GmTPR1 gene was induced by drought, low temperature, high-salt high temperature, ABA, ETH, SA. Under ABA and SA treatment for 1 h , maximal expression pattern of GmTPR1 was found. Under high-salt condition for 6 h, GmTPR1 expression level reached the maximal.Under drought treatment, GmTPR1 mRNA began to accumulate at 1 h and reached maximum at 24 h after treatment. Under low-temperature treatment, GmTPR1 mRNA began to accumulate at 1 h and reached its maximum at 12 h. Interestingly, the GmTPR1 gene was also induced by exogenous ETH and high-temperature. thereby expression of GmTPR1 gene was thought to be induced by multitudinal treatment, and it might be involved in different signal transduction pathway.5. GmTPR1 can enhance transcriptional activation activity of GmDREB5 in yeast cells YepD5 and pGADTPR1 recombine plasmid were co-transformed into yeast strains carrying the wild type DRE element, which was site before HIS3 and LacZ reporter genes in chromosome. The results showed that co-transformed yeast grew well on SD/-Ade/-His/-Leu/-Trp medium plus 20 m mol/L 3-AT, but yeast cells transforamed without pGADT7-GmTPR1 didn't grow at all on the same medium, suggesting GmTPR1 might enhance transcriptional activation activity of GmDREB5.6. Interactive site analysis between GmTPR1 and GmDREB5 proteins The full-length and different length of GmTPR1 fragments were inserted into the pGADT7 vector using site-sepecific recombinant enzyme, and co-transformed into the yeast AH109 with pGBKT7-GmDREB5. The results indicated that only the yeast transformated with the vector containing C-terminal sequencing of GmTPR1 could grow on SD/His- /Ade- /Trp- /Leu- medium, suggesting that C-terminal sequencing of GmTPR1 might play a crucial role in the interaction between GmTPR1 and GmDREB5.7. Functional analysis of GmTPR1 gene GmTPR1 gene was transformed into tobacco and Arabidopsis thaliana by Agrobacterium method approach for fuctional analysis.The results indicatied that the over-expression of GmTPR1 gene significantly improved drought and high-salt stress resistance in the two kinds of transgenic plants, and the size of parts above and under ground form the transgenic plants were longer than the corresponding parts form wild-type after stresses treatment. The Chlorophyll contents in transgenic lines were higher than in wild-type.Above results suggested GmTPR1 and GmDREB5 might interact in the nuclei of plant; C-terminal sequence of GmTPR1 might play a crucial role in the interaction between GmTPR1 and GmDREB5; GmTPR1 can enhance transcriptional activation activity of GmDREB5 in yeast cells; Expression of GmTPR1 gene was induced by multitudinal treatments and the over-expression of GmTPR1 gene significantly improved drought and high-salt stress resistance of transgenic tobacco and Arabidopsis thaliana.Ⅱ. Isolation of stress responsive genes using nuclear transportation trapNuclear proteins play vital roles in the growth and development of plants. The research on the component and the dynamic change of plant nuclear protein is essential to study on resistance-related genes of the regulation network. Current research methods are complex to operate and high cost. Therefore, it is important to build some simple and low cost techniques. In previous research, a high-efficiency nuclear transportation trap (NTT) system was constructed. In this study, the screening of wheat (Variety xiaobaimai treated under drought condition for 5 h ) cDNA library was completed using NTT system,and one (named TaABP1) of clones belonged to bZIP genes family was further studied.The major results are following:1. Identification and application of nuclear transportation trap (NTT) system In order to identify the reliability of NTT system, the one coding GmAREB with NLS (Nuclear location signal) motif was inserted into the pLEXAD vector and transformed into the yeast EGY48. The results showed that the yeast transformed with recombinant vector could survious on SD/His-/Leu- medium, suggesting that nuclear transportation trap (NTT) system is worked. We constructed wheat cDNA library and screened up 200 yeast clone using NTT system. The sequence results showed that, 29% genes among them is related to stresses response; 23% genes is related with energy and primary metabolism; The ratio of signal transduction related genes was 23%; The ratio of transport genes was 8%; And unknown protein was 17%.However, in all proteins, the most ones belonged to the type of nuclear proteins, which was accounted for 59.4%.2. Isolation and functional analysis of TaABP1 gene A novel basic leucine zipper transcription factor was isolated from wheat cDNA library using NTT system, and was named as TaABP1. Phylogenetic analysis showed that TaABP1 had higher homology with TaABI5 and TmABI5. The transcriptional activation activity analysis showed that TaABP1 did have the activation activity in yeast. The subcellular localization assay indicated that TaABP1 was localized in nucleus. Analysis of gene expression pattern indicated that the expression of TaABP1 was intensively induced by ABA, high salt, low temperature and drought. Under multitudinal treatment condition, tissue-specific expression pattern analysis indicated that TaABP1 expressed in the root, stem and leaf of wheat, and the expression quantity was decreased successively in the tissues of leaf, stem, and root. The functional analysis results indicated that TaABP1 gene improved the resistance to drought stress of transgenic tobacco significantly.3. Study on induction and differentiation of the callus from mature embroys of wheat Mature embryos of wheat as explants were cultured on the medium with different concentrations and combinations of 2,4-D and KT after different pretreatments to studyt their effects on callus induction and differentiation . It was shown that low temperature pretreatment(at 4℃) increased the calluses induction rates, Empleying this technique, regeneration rate of some genotypes was more than 30%.the results also indicated that 2,4-D concentration was related to genotypes and the addition of KT could abate the inhibition of high concentrations 2,4-D.