Cloning And Functional Study Of Candidate Transcription Factor Gene CtFRMYB1 Regulating Flavonoid Synthesis In Safflower(Carthamus Tinctorius L.)

Safflower is a plant of the compositae family.As a traditional Chinese medicine,safflower has a long medicinal history of 1800 years with the effects of promoting blood circulation and removing blood stasis.In addition,safflower is also widely used in cosmetics,oils,food.Flavonoids,the main active components of safflower,playing an important role in cardiovascular,cerebrovascular and gynecological diseases.At present,it has been reported that transcription factor MYB regulates the flavonoids synthesis in plants.The candidate transcription factor CtFRMYB1 involved in the regulation of flavonoid synthesis in safflower has been screened by our subject group,but its function is still unclear.Therefore,this study cloned candidate transcription factor gene CtFRMYB1 and investigated its function in regulating the synthesis of flavonoid in safflower.The development of this study will lay the foundation of research for elucidating the regulatory mechanism of flavonoid synthesis in safflower.Firstly,CDS of CtFRMYB1 were cloned in this study.Then,bioinformatics analysis was carried out.Secondly,the systems of protoplast isolation and transient transformation in safflower were established and the subcellular localization of CtFRMYB1 was conducted.Then,the transient expression system was used to research the regulation of CtFRMYB1 to functional genes of flavonoids synthesis.The expression vector was constructed to obtain the protein of CtFRMYB1.Finally,the interaction between the CtFRMYB1 and the promoter of flavonoids synthesis genes was analyzed by Biacore.This study provides a reference for the regulation mechanism of flavonoids synthesis in safflower.The main results of the research are as follows:1.The CDS of CtFRMYB1 were cloned.Bioinformatics analysis showed that 162 amino acids were encoded,the molecular weight of CtFRMYB1 protein was 17878.15,the isoelectric point is 4.82 and the extinction coefficient is 4470.The protein is hydrophilic,unstable and located in the nucleus.2.The effects of centrifugal force and explants on the protoplasts isolation and transformation of safflower were analyzed.The results showed that the high quality protoplasts were obtained by enzymolysis of safflower corolla tube which flowered for2 days by centrifuging at 100 g.The transformation was conducted by 110 μL PEG4000.YFP-CtFRMYB1 was constructed to perform the subcellular localization.The result showed that CtFRMYB1 was mainly expressed in the nucleus.These results indicate that the established transient protoplast system can be used for subsequent gene function verification3.Five promoters(p CtCHI2,p CtHCT5,p CtHCT12,p CtCHS3,p CtCHI1)of flavonoid synthesis genes in safflower were cloned,and three promoter activity determination vectors(Luc-p CtCHI2,Luc-p CtHCT5,Luc-p CtHCT12)were constructed.The results of activity analysis showed that CtFRMYB1 inhibited the activity of p CtHCT5 and enhanced the activities of CtCHI2 and p CtHCT12.The results of RT-PCR showed that CtFRMYB1 significantly upregulates the expression of CtC4H2,CtF3H4,CtHCT12.4.pET32-CtFRMYB1 was constructed to obtain the CtFRMYB1 protein.The result of prokaryotic expression showed that 35-40 KD protein was obtained.Twenty three MYB elements were obtained by analysis of ten promoters sequences.Finally,the result of interaction showed that CtFRMYB1 combined with “CAACCA”element of MYB.The results of this study showed that the CDS of CtFRMYB1 was 489 bp.CtFRMYB1 is a typical transcription factor,which can enhance the promoter activity of p CtHCT12 and p CtCHI2.The expression of CtC4H2,CtF3H4 and CtHCT12 genes were upregulated when overexpression of CtFRMYB1.Interaction analysis showed that CtFRMYB1 combined with MYB element "CAACCA" to regulate the expression of flavonoid synthesis genes.