Cloning And Functional Analysis Of The CtC4H1 Gene,a Key Enzyme For Flavonoids Synthesis In Safflower

Phenylpropane metabolic pathway is one of the important secondary metabolic pathways in plants.Many secondary metabolites are produced directly or indirectly by this pathway,and then flavonoid,lignin and other compounds are synthesized downstream.C4H（cinnamate4-hydroxylase）is a key enzyme in phenylpropane metabolic pathway,which regulates the biosynthesis of plant flavonoids and plant lignin.A CtC4H1 gene was screened and identified by transcriptome and qRT-PCR.The structure,physicochemical properties of CtC4H1 phylogenetic protein were described in detail.Phenotype,determination of secondary metabolite content,and expression of pathway genes were determined both in Arabidopsis thaliana with heterologous expression of CtC4H1,and safflower silenced by CtC4H1 induced by virus.The CtC4H1protein interaction network was predicted,and the subcellular localization,yeast two-hybrid（Y2H）and bimolecular fluorescence complementation（BiFC）techniques clarified the localization position and interacting proteins of the CtC4H1 protein.The difference of expression level of CtC4H1 in response to abiotic stress revealed the antioxidant activity of CtC4H1 transgenic Arabidopsis thaliana under drought stress.The specific research results are as follows:1.Genome Sequencing Two candidate CtC4H genes were identified by thermography of transcriptome data and real-time fluorescence quantitative PCR（qRT-PCR）.The expression level of CtC4H1 was always higher than that of CtC4H2,so CtC4H1 was selected for cloning and its coding region was 1518bp.Multiple sequence alignment and homology indicated that CtC4H1 gene in safflower was closely related to C4H in other species,both of which had Cytochrome P450 domain,membrane anchor region,proline rich region,threonine binding region,heme binding region and E-R-R triad domain.Meanwhile,CtC4H1 had six characteristic substrate recognition sites of CYP73A subfamily,including（SRS1,SRS2,SRS3,SRS4,SRS5and SRS6）which were closest to CYP73A subfamily.CtC4H1 protein is a hydrophilic unstable protein with a transmembrane domain and abundant phosphorylation sites.2.The Arabidopsis thaliana with heterologous expression of CtC4H1 was screened and identified by genome,and two high expression lines,OE4 and OE10,were selected from 12strains T3 transgenic Arabidopsis thaliana strains with qRT-PCR.The phenotypic statistics of wild type（WT）and two overexpressed lines（OE4 and OE10）showed that transgenic Arabidopsis thaliana had more obvious growth potential than WT.CtC4H1 transgenic Arabidopsis thaliana has larger leaves and more rosette leaves,earlier bolting time,larger stem diameter and accumulation of purple in stem tip.The content of total flavonoids and anthocyanins in CtC4H1 transgenic Arabidopsis thaliana was 1.5 times and 2.5 times higher than that in wild type.The expression of flavonoid biosynthesis genes（At PAL,At4CL,At CHS,At CHI,At F3H,At F3’H,At FLS,At DFR and At ANS）in over-expressed plants was up-regulated,At PAL gene up-regulated by 130-fold.3.In the virus-induced（VIGS）safflower CtC4H1 gene silencing experiment,the silencing efficiency of CtC4H1 plants mediated by TRV virus was tested,and Line1 and Line3 with higher silencing efficiency were selected for comparative analysis.Phenotypic observation of safflower plants before and after injection showed that the plants with transient overexpression showed a slightly better growth trend than the control,especially at the seventh day of injection.After gene silencing,the leaves of plants showed obvious yellowing and the growth of plants was inhibited,showing a trend of growth delay with the passage of time.The content of total flavonoids and anthocyanins in safflower silenced by CtC4H1 gene mediated by TRV virus were0.3-0.5 times and 0.3-0.6 times of control respectively.The expression of genes involved in the biosynthesis of flavonoids（CtPAL,CtCHS,CtCHS,CtCHI,CtF3’H,CtFLS,CtDFR and CtANS）was significantly reduced.4.The interaction network of CtC4H1 in safflower was predicted.The results showed that C4H protein was closely related to other proteins（PAL,4CL,HCT,FLS,ANS,etc.）in the pathway,the interaction between C4H protein and other proteins was possible.The localization of CtC4H1 and CtPAL1 was mainly distributed in plasma membrane and nucleus.The interaction potential between CtC4H1 and CtPAL1 was verified by yeast two-hybrid（Y2H）,and the interaction between CtC4H1 and CtPAL1 was verified by bimolecular fluorescence complementarity（BiFC）.5.The expression level of CtC4H1 gene was verified by qRT-PCR under drought,light,dark and abiotic stress,Compared with other treatments,the expression level of CtC4H1 gene increased significantly from 48 h under external drought treatment,and some phenomena indicated that CtC4H1 could regulate the response mechanism of safflower to drought stress.6.CtPAL1 and CtC4H1 overexpressed Arabidopsis strains were selected to study the role of CtPAL1 and CtC4H1 in drought stress overexpressed strains.The phenotypes of two CtC4H1overexpression lines（OE4 and OE10）and CtPAL1 overexpression lines（OE6）after 14 days of drought stress showed that the degree of drought-damaged leaves was slightly less than that of WT Arabidopsis thaliana.The results of staining with 3,3’-diaminobenzidine（DAB）and nitroblue tetrazole（NBT）showed that the accumulation of hydrogen peroxide（H₂O₂）and superoxide anion（O₂^-）in transgenic plants was less than that in control plants.After drought stress,the content of total flavonoids and anthocyanins in transgenic lines was significant,and the content of MDA in transgenic leaves was significantly lower than that in WT.Compared with wild type Arabidopsis,the activity of SOD,CAT,POD and the content of proline involved in ROS detoxification in transgenic lines induced by drought stress were more obvious than those in WT Arabidopsis.These results suggest that CtC4H1 and CtPAL1 may promote ROS scavenging in Arabidopsis thaliana by inducing the expression of flavonoid biosynthesis genes and promoting the accumulation of flavonoids and antioxidant defense system.