The Study On Cloning Of Safflower PAL4 Gene And Optimization Of The Suspension Cell Transformation System

Safflower has the effect of accelerating blood circulation to dissipate blood stasis,odynolysis,and making the meridians and collaterals unhindered,which can treat of blood stasis effectively.The main active chemical constituents of safflower are flavonoids.The biosynthesis pathway of safflower flavonoids is one of the important secondary metabolic pathways and initiates by phenylalanine ammonia-lyase?PAL?.PAL is the key enzyme catalyzed phenylpropane anabolism.At present,there have been many studies on PAL of various plants.However,there has not been a reported the homologous expression of PAL gene in safflower suspension cells.We conducted a study on a genome-wide bioinformatics analysis of the safflower PAL gene family.Candidate PAL gene sequences were obtained and a gene of phenylalanine ammonia-lyase Ct PAL4 of safflower was cloned.The genetic transformation system of safflower suspension cells was optimized,and the overexpression of Ct PAL4 gene in safflower suspension cells was preliminarily realized by agrobacterium-mediated method.This study laid a foundation for studying the function of Ct PAL4 gene in the anabolic pathway of flavonoids in safflower.This study provides a theoretical basis for the study of improving the content of flavonoids and secondary metabolism of flavonoids compounds in safflower by means of genetic engineering.1.Complete the bioinformatics analysis of the PAL gene family of safflower.Through bioinformatics analysis of the safflower PAL gene family,six safflower PAL genes were retrieved.All the members of Ct PAL1?5 gene family in safflower contain MIO domain and can catalyze phenylalanine to cinnamic acid.The number and types of cis-acting elements are different in the upstream promoter of the six safflower PAL genes.The six safflower PAL genes were clustered in the same evolutionary branch,and meanwhile they were clustered in the same branch with Arabidopsis thaliana,indicating that safflower and Arabidopsis thaliana were closely related to each other.Transcriptome analysis showed that the PAL gene family of safflower had significant differences in expression levels in different flowering stages,different germination stages of seeds and different tissues and organs.Successfully cloned a safflower PAL4gene,called Ct PAL4.Specific primers were designed according to the candidate PAL gene sequence,and Ct PAL4 gene was obtained by RT-PCR with c DNA of safflower petals as the template.Through the gradient PCR experiment,the optimal annealing temperature was 58?.The complete coding frame of Ct PAL4 was 2124 bp.2.Bioinformatics analysis of Ct PAL4 gene was completed.The results showed that the ORF of Ct PAL4 gene was 2124 bp,encoding 708 amino acids,and the enzyme molecular weight is 76.82 k Da.The three-dimensional structure of the protein encoded by Ct PAL4 gene is similar to that of the X-ray protein crystal structure of phenylalanine ammonia-lyase,which is homologous tetramer.And it has the same domain as PAL,and has the typical characteristics of PAL.Subcellular localization predicts that its position in the cell may be located in the chloroplast and cytoplasm.Phylogenetic trees show that the safflower PAL gene and Arabidopsis thaliana PAL group into one class.That means the PAL of safflower and Arabidopsis thaliana are closely related.3.The plant expression vector of Ct PAL4 gene was successfully constructed.The p CAMBIA 3301 was used as the skeleton of plant expression vector.Bgl II and Bst E II were introduced into both ends of Ct PAL4.The GUS reporter gene on the vector was replaced with the Ct PAL4 to construct the plant expression vector by double enzymolysis-connection.Plant expression vector p CAMBIA 3301-Ct PAL4 was successfully constructed and transformed into competent cells of Arabidopsis thaliana EHA105 by freeze-thaw method.4.The suspension cell genetic system of safflower was established and optimized.The overexpression of Ct PAL4 gene in suspension cells was preliminarily realized.The cotyledon explants of sterile seedlings from dark culture of safflower seeds was induced into callus,which was proliferated and screened.Screening was performed using carbenicillin?75 mg/L?-cephalosporin?100 mg/L?-glyphosate?1 mg/L?.Agrobacteria-mediated genetic transformation conditions:suspension OD₆₀₀=0.5,engineered agrobacterium OD₆₀₀=0.8,infected for 15 min at a vacuum of 10 mba,and cultured for 4 days to obtain transgenic safflower callus.The safflower callus of transgenic was subjected to slight mechanical vibration to form uniform suspension cells of transgenic safflower.GUS histochemical analysis showed that more than 70%of the callus was blue.PCR was used to detect safflower suspension cells with Ct PAL4 gene,and the band position of the target gene was consistent with the theoretical position.It suggests that Ct PAL4 is integrated into the safflower suspension cell genome.To sum up,the bioinformatics analysis of safflower PAL gene family was completed.Ct PAL4 gene was successfully cloned,and p CAMBIA 3301-Ct PAL4 plant expression vector was constructed.The suspension cell genetic system of safflower was established and optimized.The homologous expression of Ct PAL4 gene in safflower suspension cells was realized.The successful introduction and expression of the Ct PAL4 in safflower suspension cells laid a theoretical and practical foundation for the study of the functions of Ct PAL4 and other key anabolic enzyme genes in secondary metabolites and the regulation of secondary metabolism and synthesis of flavonoids in safflower.