Cloning And Functional Analysis Of Salt Tolerance Of The Transcription Factor Gene Cptaf10 From Chimonanthus Praecox

Transcription factor is the DNA-binding protein which specifically interacted with cis-acting elements in the promoter region of eukaryotic gene. TAFs,the TATA box-binding protein associated factors, is a class of the coupling factor of eukaryotic TATA-binding protein (TBP). TAF10, also known as TAFⅡ30, is one of the many TAFs involved in the formation of the TFIID.The gene TAF10 regulated the expression of genes related to cell cycle progression and plant morphology.On the basis of a cDNA library and EST analysis, we obtained a transcription factor gene CpTAF10 in Chimonanthus praecox by random cloning and sequencing.The real-time quantitative PCR and the plant expression were used to analyze its potential biological functions.The main results are as follows:1. Bioinformatics analysis of CpTAF10 gene and CpTAF10 proteinThe full length of CpTAF10 cDNA sequence is 698bp, which contains a 405bp open reading frame. This gene encoded a 134 amino acid with no intron. The 134 amino acids of CpTAF10 protein contains 40 hydrophobic amino acids and 35 polar amino acids. The encoded protein contains 40 hydrophobic amino acids and 35 polar amino acidswith theoretical molecular weight of 15.21 KD, isoelectric point p value of 5.123.The secondary structure mainly was composed of random coil (47.76%)、the a helix (47.01%) and extension chain (5.22%).2. Real-time quantitative PCR analysis of the CpTAF10 transcriptionAccording to expression studies by real-time quantitative PCR, CpTAF10 was constitutively expressed in different organs under normal conditions, which was highly expressed in mature leaves than other organs. CpTAF10 transcripts were detected and more abundant in petals during flower opening.CpTAF10 was strongly induced under the the pressure of various abiotic stresses such as high temperature, low temperature, salinity, ABA and heavy metel. All these transcriptional expression analysis results suggested that CpTAF10 may play a significant role in plant development such as the flavor formation, stamen development and abiotic stress responses.3. Overexpression CpTAF10 gene in tobaccoPlant expression vector Containing the Chimonanthus transcription factor gene CpTAF10 was transformed into tobacco by agrobacterium-mediated transfor mation.After GUS staining、the Km+ resistance selection detection、PCR detecti-on and RT-PCR detection,we obtained 15 transgenic plants.After salt stress on seedlings from tissue culture and cultivation, we found that transgenic plants salt tolerance was strong compared with wild control. So we determined preliminaryly that CpTAF10 gene mattered with the salt tolerance.