Preliminary Functional Analysis Of Gene CpTAF9 And Gene CpTAF10 From Chimonanthus Praecox(L.)Link

Transcription factor which named trans-acting factor, is the DNA-binding protein which specifically interacted with cis-acting elements in the promoter region of eukaryotic gene. TAFs (TATA box-binding protein associated factors) that TATA-box binding protein-associated factor, is a coupling factor belongs to a class of eukaryotic TATA-binding protein (TBP). Both TAF9 and TAF10 are TAFs family members.On the basis of full-length CpTAF9 gene, which has been cloned by the laboratory, we analyze the bioinformatics of CpTAF9 protein, and construct the plant over-expression vector containing the gene of CpTAF9, then transform the vector into Arabidopsis wild type. After treatments on seeds or seedlings of transgenic Arabidopsis with salt and heat stress, we get the preliminary analysis of CpTAF9 gene function. Moreover, we transfer the over-expression vector of CpTAF10 gene into Arabidopsis wild type. After treatments on seeds and seedings of transgeic Arabidopsis with salt stress, a preliminary analysis of CpTAF10 gene function has been conducted. The main findings are as follows:1. Bioinformatics analysis of CpTAF9 gene and its encoding proteinThe full length of CpTAF9 gene is 870bp, which contains a 603bp open reading frame. This gene encodes 134 amino acids with no introns. Multiple sequence alignments show that CpTAF9 protein has a high homology with homologous proteins. The cluster analysis shows CpTAF9 protein and the homologous protein of Cucumber are in the same branch.2. Real-time quantitative PCR analysis of the CpTAF9 gene expression at transcription levelReal-time PCR analysis showed that the expression of transcripts in mature leaves up to the amount, followed by young leaves, and there has significant difference between the two, indicating that the gene expression has specific in developmental stage. Speculate that the gene function is associated with leaf morphogenesis. CpTAF9 gene expression is different after different abiotic stresses in leaves. When subjected to high temperature or salt stress, the expression was significantly increased, indicating that this gene plays a role in tissue development and stress response process. 3. Overexpression CpTAF9 gene and CpTAF10 gene in ArabidopsisWe construct the plant expression vector pCAMS1A2301g-CpTAF9, and then transform it into Arabidopsis thaliana by agrobacterium-mediated transformation. We screen the Arabidopsis seeds by ueing MS resistance medium, till obtaining T2 generation transgenic plants. After GUS staining and RT-RCR detection, we got seven transgenic plants.After treated on transgenic plants for seeds germination and seedlings growth with salt stress, we found that transgenic plants were stronger than Arabidopsis wild type; after treated with heat stress, the transgenic plants were found it has stronger heat resistance than the Arabidopsis wild type. We preliminary determined CpTAF9 gene can improve salt tolerance and heat resistance in Arabidopsis.After treated transgenic Arabidopsis on seeds germination and seedlings growth with salt stress, we found that transgenic Arabidopsis were stronger than Arabidopsis wild type. So we preliminary determined that CpTAF10 gene was associated with the salt tolerance.