Molecular Cloning And Characterization Of Chalcone Isomerase Genes In Freesia Hybrida

Freesia hybrida is a good plant material to study the secondary metabolism of flower color because of its various color.Flavonoids are one of the main factors that form flower color.Flavonoids fulfill many biological function,and its synthesis are affected by many factors,including structural genes,regulatory genes,environmental factors and so on.Then these factors can modulate flower color through changing the kinds and contents of flavonoids.Chalcone isomerase is the key enzyme in the second step of flavonoids biosynthesis pathway.It has been reported that chalcone isomerase plays a critical role in flower color metabolism as well as the biosynthesis of flavonoids.In this study,we successfully cloned chalcone isomerase gene family of F.hybrida(FhCHIs)and analysed their difference in function by prokaryotic and eukaryotic expression.It's hoped to make sure which FhCHIs directly participate in the synthesis of flavonoids in F.hybrida.This research may lay a good foundation for modifying the color of flower by plant genetic engineering.The main results were as follows:1.FhCHI gene family were cloned and analysed.Based on transcriptome information of F.hybrida and fragment information from degenerate PCR,a total of 5 FhCHIs were cloned including their cDNA and gDNA.The Open Reading Frame of FhCHI1 ~ FhCHI5 were 687?771?771?630?834 bp long,encoding proteins with 228?256?256?209?277 amino acids respectively.FhCHIs shared the highest homology to CHIs from Tulipa fosteriana,Narcissus tazetta,Phoenix dactylifera,Iris x hollandica,Narcissus tazetta.There were five conserved active site amino acid residues in the amino acid sequences of FhCHI1,FhCHI2 and FhCHI5;but they were absent in FhCHI3 and FhCHI4.Phylogenetic analysis showed FhCHI1,FhCHI2 and FhCHI5 belong to type?CHI,while FhCHI3 and FhCHI4 belong to type ? and type ? CHI respectively.2.Expression patterns of FhCHIs.During flower development of red F.hybrida,the highest expression level of FhCHI1,FhCHI3 and FhCHI4 were observed in slightly pigmented buds;as for FhCHI2 and FhCHI5,their expression level was lowest in fully pigmented flowers before complete opening,and then rised again in fully opened flowers.The transcription of FhCHIs were clearly tissue specific,and all of them were high in torus and petal;while the expression level of FhCHI1 and FhCHI3 were lower than other FhCHIs.3.Expression and purification of Fh CHIs recombinant proteins and assay of their activity.Firstly,we successfully constructed the prokaryotic expression vectors pET28a-FhCHIs;and they were introduced into E.coli BL21;and then prokaryotic expression recombinant proteinswere harvested by culturing at low temperature and purifying;at last,enzyme activity of FhCHIs were tested by HPLC.The result indicated that FhCHI2 and FhCHI5 can catalyze the formation of naringenin,while FhCHI3 and FhCHI4 can't.4.Heterologous expression of FhCHIs.Firstly,we constructed eukaryotic expression vectors pBI121-FhCHIs;next,transferred them into Arabidopsis mutant tt5,and then screened for positive transgenetic plants.The result showed that transgenetic plants of FhCHI1,FhCHI2 and FhCHI5 recovered the purple phenotype in hypocotyls and brown pigmentation in seed coats,in the meanwhile,HPLC analyses revealed that these transgenetic plants could restore the biosynthesis of anthocyanins and flavonols;while transgenetic plants of FhCHI3 and FhCHI4 could't.The results indicated that FhCHI1,FhCHI2 and FhCHI5 played an essential role in flavonoids biosynthesis pathway in F.hybrida.