Cloning Of CtCYP71A1 Gene From Safflower And Its Regulation On Lignin Biosynthesis

Safflower is a traditional Chinese herb that has some medicinal and economic values.It is widely grown in Xinjiang,Sichuan and other places and has certain drought tolerance.At present,studies on the drought tolerance mechanism are less.Cytochrome P450（CYP450s）is the largest enzyme protein family in higher plants,which plays a very important role in the basic and secondary metabolism of plants.Cytochrome P450（CYP450s）plays a wide and complex role in catalyzed reaction types.However,the function of CtCYP71A gene family in Safflower has not been analyzed yet.In this study,bioinformatics analysis was conducted on CtCYP71A subfamily genes based on sequencing data of Safflower genome,and phylogenetic analysis was conducted on 16screened CtCYP71A subfamily genes,and the conserved motif,gene structure and cis-acting elements of the subfamily genes were predicted.RNA sequencing（RNA-Se Q）and quantitative fluorescence PCR（qRT-PCR）were used to analyze the expression patterns of different tissues of Safflower.CtCYP71A1,a member of the CtCYP71A subfamily of Safflower,was cloned and bioinformatic analysis and subcellular localization analysis were performed.The change of the expression pattern of CYP71A1 gene under different treatments was analyzed,and the function of CtCYP71A1 in lignin synthesis and drought stress was preliminatively investigated by using transgenic Arabidopsis Thaliana and transient transformed safflower.The main research achievements are as follows:1.Sixteen CtCYP71A genes were screened based on the Safflower genome database.Phylogenetic analysis showed that the CtCYP71A family genes clustered in the same branch as the Arabidopsis CYP71 family type A,and amino acid structure and gene structure analysis indicated that the subfamily members had high structural conservation.Promoter analysis showed that this family gene had response elements such as drought,methyl jasmonate（MEJA）,salicylic acid（SA）and gibberellic acid（GA3）,and RNA-seq data and qRT-PCR results showed that CtCYP71A subfamily gene had tissue expression specificity.Combined with bioinformatics and expression analysis results of different tissues,it was found that CtCYP71A1 gene of CtCYP71A subfamily was specifically expressed in root tissues of Safflower.2.CtCYP71A1 gene was cloned from Safflower root,and the coding region sequence was1287 bp.The secondary structure and tertiary structure of CtCYP71A1 protein were predicted.It was found that the proportions ofα-helix,β-angle,irregular curling and extended chain were46.96%,6.54%,31.78%and 14.72%,respectively.The tertiary structure GMQE value was 0.67,indicating that the protein structure was stable.The comparison of CtCYP71A1 amino acid sequences in different species indicates that CtCYP71A1 protein sequence also contains the typical P450 protein conserved domain and has high homology with other species.3.The plant expression vector of CtCYP71A1::GFP fusion protein Pcambia131-Ctcyp71A1 was constructed and expressed in the leaves of tobacco Ben.The fluorescence signal was detected by laser confocal microscopy.Four abiotic stress treatments were carried out on wild-type safflower.The results showed that the expression of CtCYP71A1 gene was the highest at 48 h under polyethylene glycol（PEG6000）stress,the highest at 12 h under abscisic acid（ABA）stress,and then began to decrease.The expression of CtCYP71A1 reached the maximum at 24 h GA3 stress and 3h SA stress,and then decreased,indicating that CtCYP71A1 was the most responsive to drought stress of Safflower.4.By infecting inflorescence,the highly expressed strains OE-3 and OE-6 of Arabidopsis Thaliana were obtained.Compared with wild type（WT）and mutant（Mut）,the lignin content of transgenic Arabidopsis thaliana was significantly increased by 2 times.The results of PEG simulation drought in the medium showed that the phenotype was the most obvious under10%PEG treatment.The root length of the transgenic strain was significantly longer than that of the wild type,while that of the mutant was on the contrary.The roots of the transgenic strain were stained with diaminobenzidine（DAB）and nitrotetrazazole blue（NBT）chloride.It was found that the accumulation of hydrogen peroxide（H₂O₂）and superoxide anion（O₂^-）was more in mutant strains,while the content of H₂O₂ and O₂^-was less in transgenic strains.The results of soil drought stress showed that after 15 days of treatment,the growth state and fresh weight of transgenic strains were also significantly better than those of the mutant and wild type materials,and the lignin content in plant roots was 0.5 times higher than that of the control group,and the contents of malondialdehyde（MDA）,O₂^-and H₂O₂ were significantly lower than that of the mutant and wild type.Proline（Pro）content increased significantly.5.Using VIGS technique,vacuum pump infection was carried out on safflower plants at germination stage.The expression of CtCYP71A1 gene was analyzed by qRT-PCR.Three Safflower plants with CtCYP71A1 gene silencing were obtained successfully.After 20 days of soil drought,phenotypic and physiological indexes of pTRV2-CtCYP71A1 strains were detected.Compared with no-load plants,pTRV2-CtCYP71A1 strains had weaker growth,lower fresh weight and lignin content,significantly increased MDA,O₂^-and H₂O₂ contents,and significantly decreased proline content.The results indicated that CtCYP71A1 gene promoted lignin accumulation in safflower root.