Cloning, Expression Analysis And Functional Verification Of Transcription Factors Genes Ct Dof In Safflower

Safflower is a kind of herbaceous plant of Compositae genus.At present,there are about 25 species of safflower plants in the world,while in China,there is only safflower.Safflower has strong ability of cold resistance and drought resistance,mainly planted in China's Henan,Xinjiang,Sichuan,Zhejiang and other regions.Transcription factor Dof is a kind of plant-specific transcription factor,and it not only can respond to light response,participate in metabolic regulation to affect the seed germination and maturation,but also can participate in the anti-stress reaction to improve the resistance of plants,etc.It plays an important role in the process of plant growth and development.In this study,safflower was acted as the mainly experimental material.The expression of CtDof genes was analyzed by real-time fluorescence quantitative PCR.The plant over-expression vector of CtDof1 gene was constructed and successfully transferred into Arabidopsis thaliana to study its function.The main results are as follows:1.The expression of Dof gene family was analyzed by real-time fluorescence quantitative PCR in different tissues of safflower and parts of safflower seed at different developmental stages.The results showed that among the different tissues and safflower seed at different developmental stages,the expression of Unigene100791 gene was the highest in safflower Dof gene family,especially in mature seeds,the relative expression of Unigene100791 gene was 12.2 times of Unigene39146 gene,2.2 times of Unigene41817 gene,1.8 times of Unigene71654 gene,3.9 times of Unigene97057 gene;in seed development at the 16 day,of the relative expression of Unigene100791 gene was the highest,which was 10.7 times of Unigene39146 gene,2.1 times of Unigene41817 and Unigene71654 gene,0.2 times of Unigene97057 gene.It wassuggested that the expression of Unigene 100791 gene had different tissue specificity in different tissues of safflower and seeds at different developmental stages.In this study,the highly expressed Unigene100791 gene was used as the main research object,and the follow-up study was carried out.2.Using RT-PCR,successfully cloned the mid-fragment of CtDof1 gene,which proved the gene was present in safflower,and also confirmed the accuracy of transcriptome sequencing.Using RT-PCR and RACE,the CtDof1 gene was isolated from safflower seeds for the first time.The full-length cDNA sequence of CtDof1 gene was analyzed by bioinformatics.The full length of CtDof1 gene is 1274 bp,and its ORF is 981 bp,totally encoding 326 amino acids.3.The over-expression vector of CtDof1 gene was constructed and successfully transferred 14 into Arabidopsis.The seeds of transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana were treated with different concentrations of salt.The results showed that the transgenic Arabidopsis showed sensitivity to salt stress.4.The over expression vector of pBasta-CtDof1 was transferred into wild-type safflower,and 7 transgenic safflowers were identified by screening.5.The prokaryotic expression vector of CtDof1 was constructed.The recombinant plasmid pEASY-Blunt E1-CtDof1 was successfully transformed into E.coli BL21(DE3).After 6 hours of induction,the fusion protein was efficiently expressed in E.coli BL21(DE3)with the IPTG 1mmoL/L at 37 ?,which laid a foundation for the subsequent purification of the protein.