Identification Of The Safflower AP2/ERF Gene Family And Functional Research Of CtDREB52

The AP2/ERF gene family plays a key role in plant growth and development,biosynthesis,and hormone response.In this study,bioinformatics analysis of the CtAP2/ERF family genes of safflower was performed,and a CtDREB52 gene with similar expression levels and metabolic trends of flavonoid accumulation at different flowering stages was screened in combination with transcriptomic data.The subcellular localization technique was used to analyze the location of CtDREB52 in cells,explored the correlation between the transcription level of this gene and flavonoid accumulation in transgenic plants by transient transformation technique and HPLC technique,and clarified the protein interactions between CtDREB52 and key enzymes of flavonoid biosynthesis by the Yeast-two-hybrid technique,so as to clarify the regulatory role of CtDREB52 gene in safflower flavonoid biosynthesis.The expression level of CtDREB52 under abiotic stress was analyzed to investigate the effect of CtDREB52 on flavonoid biosynthesis and the response to abiotic stress.This topic laid the foundation for further research on the biosynthesis mechanism of safflower flavonoids.The specific results of the study are as follows:（1）A total of 187 complete AP2/ERF genes were identified in the safflower genome database,bioinformatics analysis of the safflower CtAP2/ERF family genes,based on sequence similarity and the number of conserved AP2 structural domains,classified the safflower AP2/ERF family into four subfamilies:AP2,ERF,DREB,and RAV.phylogenetic tree analysis showed that the Arabidopsis and safflower The phylogenetic tree analysis showed that the AP2/ERF families of Arabidopsis and safflower have some similarity at the evolutionary level.Most of the subfamily members have similar motif composition,indicating that AP2/ERF proteins in the same subfamily are functionally similar.（2）Combined with safflower transcriptome data,nine CtAP2/ERFs genes that expressed similar flavonoid accumulation and metabolism trends at different flowering stages were selected,and quantitative analysis of CtAP2/ERF genes at four flowering stages was performed by q RT-PCR.The results of q RT-PCR showed that the expression level of CtDREB52 gene was consistent with the accumulation rate of flavonoids in safflower,and at flowering highly expressed.A CtDREB52 gene with a coding region of 894 bp was successfully cloned,and the subcellular localization results showed that the CtDREB52 protein was localized in the nucleus.（3）Transient transformation of safflower showed that transient overexpression of CtDREB52 plants exhibited an earlier flowering phenotype than the control group,and transient overexpression of CtDREB52 upregulated the expression of key enzymes of the flavonoid metabolic pathway（CtCHI,CtFLS,CtMYB,CtF3’H）genes and promoted flavonoid accumulation,while transient silencing of CtDREB52 resulted in the opposite.（4）The results of Yeast-two-hybrid validation showed that CtDREB52 protein has protein interactions with CtFLS,CtF3’H and CtDFR,key genes for flavonoid synthesis.Bi FC experiments showed that CtDREB52 protein and CtMYB protein act in the nucleus,and CtDREB52 protein and CtFLS protein,CtF3’H protein and CtDFR protein act in the nucleus and cell membrane.（5）The expression levels of CtDREB52 gene were verified by q RT-PCR under different hormonal and abiotic stress conditions.The results showed that the transcript levels of CtDREB52 responded to biotic and abiotic stresses making cycle-adaptive responses.UV-B treatment of transiently overexpressed CtDREB52 plants showed that the treatment significantly induced the expression of CtDREB52 and up-regulated UV-B-induced expression of CtDFR,CtFLS,CtF3’H and CtMYB and accumulated more flavonoid content and CAT content and less H₂O₂content compared with the control.UV-B treatment transiently silenced CtDREB52 plants showed the opposite results.It indicates that CtDREB52 can improve the resistance of safflower to UV damage by regulating flavonoid and hydrogen peroxide contents.