Cloning And Genetic Transformation Of Tobacco Of DREB Transcription Factor From Iris Typhifolia Kitag

Iris is one of the largest genera of monocots Iridaceae，Iris typhifolia Kitag. is a perennial herb of Iris, China has abundant resources of wild Iris,these wildlife resources are rich in good genes and can be able to provide resources for improved varieties and germplasm.In this study,we took the total RNA of Iris typhifolia Kitag. leaves as a template,cloned some encoding 280 amino acid sequence of DREB gene protein by homologous cloning technology,named ItDREB,we analysed the biological function of genes by transgenic technology.Real-time PCR analysis showed that ItDREB gene from Iris typhifolia Kitag. seedlings can be induced by drought and high salinity expression,which play a role in the drought and high salinity signaling pathways.The main results of this study are as follows:1.DREB gene isolated from Iris typhifolia Kitag. belonging AP2 gene family, with the AP2 conserved domains.The alignment results showed that:ItDREB has a high homology with AoDREB^ CiDREB and TaDREB,speculate that their function is similar.2.Analysing the characteristic of tissue-specific expression and expression under different stress by RT-PCR method, ItDREB have expression at different levels in various tissues, have no specific tissue expression. ItDREB can be induced significantly under drought and high salt conditions,and can only be a weak expression under low temperature.3.We constructed the vector pBI121-DREB,this vector was converted into wild tobacco by Agrobacterium-mediated transformation of leaf discs and obtained transgenic tobacco,PCR results confirmed that the exogenous gene has been successfully integrated into the genome of tobacco.4.We constructed the vector pBI121-DREB-GFP,and the GFP fusion protein pBI121-DREB-GFP was bombarded into the fresh onion epidermal cells by gene gun, finally got the conclusion that GFP fusion protein pBI121-DREB-GFP expressed in the cytoplasm of onion epidermis.