Molecular Cloning And Characterization Of Chalcone Synthase And Chalcone Isomerase Genes In Clivia Miniata

Clivia miniata is a kind of flower with both foliage and flower viewing characteristics.As the city flower of Changchun,it has a higher economic value.Its flower is large and has a long flowering period but a single color,most of which is orange.It has severely restricted the growth of its ornamental value and economic value.Therefore,it is particularly important to study the mechanism of flower color formation in order to improve its ornamental value.The various flower colors of plants are mainly determined by flavonoids.Chalcone Synthase(CHS)and Chalcone Isomerase(CHI)are the key enzymes in the flavonoid biosynthesis pathway,which play an important role in the synthesis of anthocyaninsThis experiment used Clivia miniata as the test material,the full-length c DNA sequences of CHS and CHI genes were cloned by PCR from the transcription database of Clivia miniata;Bioinformatics methods were used to analyze the basic information of Cm CHSs and Cm CHIs genes to predict their structure and function;The RT-PCR method was used to analyze the temporal and spatial specificity expression of Cm CHSs and Cm CHIs genes to explore their accumulation of flavonoids in plants;The prokaryotic expression vector p ET-32-Cm CHSs/Cm CHIs were constructed,and the recombinant proteins were prepared for in vitro enzyme activity determination;The eukaryotic expression vector p BI121-Cm CHIs were constructed and transformed into Arabidopsis mutants to explore the recovery of phenotype and anthocyanin content;The bimolecular fluorescence complementation method and the Gal4:GUS system were used to verify whether the Cm CHI family proteins can interact.In this study,by exploring the bioinformatics,gene expression,enzyme activity and function of Cm CHSs and Cm CHIs genes,to study the structure and function of chalcone synthase and chalcone isomerase in Clivia miniata,it laid a theoretical foundation for the genetic transformation and flower color improvement of Clivia miniata.The main findings are as follows:1.Bioinformatics analysis(1)Through local BLAST,four potential chalcone synthase genes and three potential chalcone isomerase genes were selected from the Clivia miniata transcriptome database,named Cm CHS1,Cm CHS2,Cm CHS3,Cm CHS4,and Cm CHI1,Cm CHI2,Cm CHI3 were cloned with the full-length ORF(open reading frame),and compared with NCBI data,it was found that they have high homology with chalcone synthase and chalcone isomerase genes in other species.(2)Amino acid sequence alignment and phylogenetic tree analysis suggest that Cm CHS1,Cm CHS2,Cm CHS3,and Cm CHS4 are clustered with CHS in other species and have typical CHS conserved domains;Cm CHI1,Cm CHI2 are clustered with type I CHI in other species.It has the typical conserved domains of CHI.Cm CHI3 and type IV CHI are clustered together.They do not have CHI amino acid catalytic sites and may not have the function of CHI.(3)Construction of the recombinant vector GFP-Cm CHSs/Cm CHIs for subcellular localization the results showed that the GFP emits green fluorescence in the cytoplasm.2.The expression of Cm CHS and Cm CHI gene family has temporal and spatial specificity.According to the full-length c DNA sequence of Cm CHS and Cm CHI genes,specific primers were designed.The RT-PCR method was used to analyze the expression levels of Cm CHS and Cm CHI genes in the main stained tissues and different flowering periods.The results showed that the expression levels of Cm CHS and Cm CHI genes were highly in red tissues and lower in green tissues.In the four stages of flower development,most of them were expressed at the later stage of development and showed a positive correlation trend with the content of anthocyanin.3.In vitro enzyme activity determination of Cm CHS and Cm CHI(1)The prokaryotic expression vector p ET-32-Cm CHSs were successfully constructed for induce recombinant protein,and the in vitro enzyme activity test results showed that Cm CHS1-Cm CHS4 can catalyze the formation of naringenin chalcone from coumaroyl coenzyme A and malonyl coenzyme A,And all of the Cm CHSs have the function of chalcone synthase.(2)The prokaryotic expression vector p ET-32-Cm CHIs were successfully constructed to induce recombinant protein.The in vitro enzyme activity test results showed that Cm CHI1 and Cm CHI2 can catalyze the substrate naringenin chalcone to produce naringenin,means they have chalcone isomers activity,while Cm CHI3 can only react spontaneously without catalysis.4.Transgenic function verification of Cm CHIsThe eukaryotic expression vectors were successfully constructed and the recombinant plasmid p BI121-Cm CHIs(GV3101)was transformed into the Arabidopsis mutant.The phenotype and seed coat color of Cm CHI1/I2 transgenic Arabidopsis thaliana in T3 generation were similar to those of wild type,but the phenotype and seed coat color of Cm CHI3 transgenic Arabidopsis thaliana did not change.It proves that Cm CHI1/I2 genes have the function of chalcone isomerase,which is related to flower color formation.5.Cm CHIs protein interactionThe interaction of the three proteins of Cm CHIs were verified by bimolecular fluorescence complementation and Gal4:GUS system experiments.The results showed that Cm CHI1/I2/I3 can interact with each other,and Cm CHI3 may act as an enhancer to enhance the protein activity of anthocyanin synthesis genes.