System Analysis Of DOF Gene Family In Jilin Panax Ginseng And Preliminary Functional Analysis Of PgDof14-1 And PgDof26-5

Panax ginseng C.A.Meyer is a perennial herbaceous plant of the Panax family of Araliaceae.DNA binding with one finger(Dof)protein is a special transcription factor in plants.To explore the function of Dof gene in ginseng.Based on the transcriptome database of Jilin ginseng,which generated 248,993 transcript unigenes,75 Dof transcripts were screened in Jilin ginseng and named PgDofs.Among them,54 PgDofs have complete open reading frames and conserved domains.By bioinformatics analysis and correlation analysis,two genes,i.e.PgDof14-1 and PgDof26-5,were highly correlated to saponin content.After cloned these two genes from Jilin ginseng.And Arabidopsis thaliana and hairy roots they were successfully transformed to Arabidopsis thaliana and ginseng hairy roots by Agrobacterium tumefaciens and Agrobacterium rhizogenes-mediated transformation,respectively.Overall,this study could lay a foundation for the functional verification of PgDof14-1 and PgDof26-5.The research results were as follows:1.The average length of 75 PgDofs was between 200 bp and 2400 bp.2.Dof protein were generally involved in three aspects of plants,including biological processes,molecular functions and cellular components.At the level 2,the main functions are concentrated in eight functional categories: Cellular process(GO:0009987),Metabolic process(GO: 0008152),Biological regulation(GO: 0065007),Regulation of biological process(GO: 0050789),Binding(GO: 0005488),Cells(GO: 0005623),Organelles(GO: 0043226),and Cell parts(GO: 0044464).3.The results of phylogenetic tree analysis showed that all PgDof genes had Zf-Dof motif,indicating that the domain was evolutionarily conservative,and PgDofs were divided into five groups according to sequence similarity.4.The expression patterns of PgDofs in 14 tissues of Jilin ginseng,42 farm cultivar and four different old-year roots showed expression of PgDofs had certain tissue specificity and temporal specificity.By constructing an inter-genetic interaction network,the inter-gene interaction model was further determined.The expression activities of the PgDof genes were not independent,but affect each other's metabolic processes involved in the plant.5.Correlation analysis with saponin content showed that 32 PgDof genes were significantly associated with the ginsenoside content.By the correlation analysis between PgDof genes and the key enzyme genes in the ginsenoside synthesis pathway,46 genes were found to be significantly associated with key enzyme genes.Besides,24 PgDof genes were significantly associated with both key enzyme genes and saponin content.6.Network analysis of 24 PgDof genes and key enzyme genes showed that PgDof14-1 was always linearly linked to CYP716A52v2-3 gene under the condition that P-value was gradually strict.It is speculated that this gene may directly or indirectly participate in the synthesis of ginsenosides.7.The results of SNP analysis showed that among the 54 PgDofs,24 genes had 360 SNPs.Among them,18 genes,which had 165 SNPs,had significant effects on ginsenoside content.Besides,8 genes were significantly correlated with both saponin content and key enzyme gene expression,i.e.PgDof25,PgDof28,PgDof24,PgDof26-3,PgDof17-2,PgDof26-5,PgDof29-1 and PgDof20.8.According to the results of Heatmap and Network and SNP,the candidate genes were identified.and the ginseng hair root was used as the material for cold stress treatment,and real-time PCR was performed.The results showed that the PgDof14-1 gene was up-regulated and the PgDof26-5 gene was down-regulated.It is speculated that the two genes may be related to responsing to cold tolerance of ginseng.9.The PgDof14-1 and PgDof26-5 genes were successfully cloned from ginseng.Then,they were inserted into the plant expression vector pBI121,and further transformed to Arabidopsis thaliana by Agrobacterium tumefaciens.Under low temperature,the content of cholorophyll in positive Arabidopsis thaliana was significantly higher than the wild type Arabidopsis thaliana.10.Therefore,it is supposed that both PgDof14-1 gene and PgDof26-5 gene were possessed cold resistance function in ginseng.