| As a new type of oil crops,safflower has become a research hotspot in recent years.Safflower oil is rich in unsaturated fatty acids,so safflower oil is widely used in food,medicine and industry.Especially in the development of alternative energy sources,safflower oil,as one of the potential alternative energy sources for petroleum,has wide application prospects.Dof transcription factors are a class of proteins unique to plants,and the existence of Dof transcription factors has not been found in yeast and nematodes.Members of the Dof family play an important role in plant growth,development and physiological processes.More and more Dof transcription factors are involved in the regulation of plant seed oil synthesis,but the gene information and functions of Dof transcription factors in safflower have not been reported.Based on the safflower genome database,this study screened and identified members of the safflower Dof family,analyzed the spatiotemporal specific expression patterns of the safflower Dof family genes,and screened out the CtDof2 gene that is highly related to seed development and oil accumulation.A yeast expression vector was constructed to transform Pichia pastoris X33 cells and analyse the effect of the CtDof2 gene on yeast lipid and fatty acid fractions.A plant expression vector was constructed to obtain stable T3 generation overexpression of Arabidopsis thaliana and mutants complement seeds to clarify the function of the CtDof2 gene in Arabidopsis thaliana.The results of the study are as follows:1.Based on the safflower genome database,33 safflower Dof genes were screened and named as CtDof1-CtDof33.According to phylogenetic analysis,the Dof gene family of safflower was divided into 6 subfamilies(A,B,C,D,E and F),with the largest molecular weight of 46.07 k Da and the smallest of 17.39 k Da.The GRAVY values of all CtDofs were negative value,indicating that CtDof family proteins are hydrophilic proteins.Conserved motif analysis found 11 Motifs,and Motif1 was a conserved Dof domain.24 CtDof genes were co-annotated into three processes of MF,CC and BP.The results showed that the CtDof gene promoter has light-responsive,hormone-responsive,low-temperature stress,seed-specific and endosperm-specific expression elements.2.The q RT-PCR results showed that CtDof24 was highly expressed in flowers and roots,CtDof13 was highly expressed in leaves,and CtDof2 was expressed at the highest level in seeds.The expression of CtDof2 gene first increased and then decreased in different developmental stages of seeds.3.Yeast expression vectors were constructed and yeast cells were transformed.It was found that the lipid content of yeast transfected with the CtDof2 gene was increased and the number of liposomes and fluorescence intensity were greater than those of wild-type yeast and yeast transfected with the empty vector.Analysis of fatty acid content showed that the transgenic yeast had increased oleic acid content and decreased stearic acid content.4.Infect wild-type Arabidopsis thaliana and mutant Arabidopsis thaliana by floral dip method,and obtain T3 generation high expression line after screening and identification.Comparing the traits of different Arabidopsis strains,it was found that overexpressing CtDof2 gene Arabidopsis thaliana had significantly increased1000-grain weight,and the 1000-grain weight of mutant Arabidopsis thaliana seeds was lower than that of wild-type Arabidopsis thaliana seeds.The oil content of Arabidopsis thaliana seeds overexpressing the CtDof2 gene was significantly increased and the contents of C16:0,C18:1 and C20:1 fatty acids were increased,and the contents of C18:2 and C18:3 fatty acids were decreased.5.Through the expression analysis of key genes in the lipid synthesis pathway,it was found that CtDof2 gene positively regulates the expression levels of DGAT1,ACP1,ACC1 and SSS1 genes,and negatively regulates the expression levels of FAD3 and AGPase-S1 genes,thereby affecting the content of fatty acids. |
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