Molecular Cloning And Characterization Of Chalcone Synthase And Flavanone 3-hydroxylase In Clivia Miniata

Clivia miniata is a kind of perennial herbaceous plant that belongs to clivia genus of Amaryllidaceae family.It originates from Southern Africa after introduced into China by Germany and Japan.It was an important indoor ornamental plant,larger flowers and long flowering,and has been named city flower of changchun.However,its ornamental and economic value has been influened because of its single color of flower.It is very meaningful of investigate C.miniata flower color formation mechanism.The plant flower color is mainly determined by the amount and kind of flavonoids,carotenoids and betalains.Among them,flavonoid is the main factor to affect the flower color.Both chalcone synthase and flavanone-3-hydroxylase are the key enzymes of flavonoid pathway.A large number of studies have shown that chalcone synthase and flavanone-3-hydroxylase plays an very important role in the formation of plant flower color.In this study,The cDNA clone encoding CHS and F3 H were amplified by RACE-PCR techniques from petals of C.miniata.Sequence analysis showed that the CHS cDNA is 1173 bp long encoding 390 amino and F3 H cDNA is 1128 bp long encoding 375 amino.The bioinformatics analysis,construction of eukaryotic expression vector and characterization of gene function by Arabidopsis transgenic and construction of prokaryotic expression vector and characterization of protein function by enzyme reaction.This study would lay a good foundation for improvement and research of flower color.The main results as follows:1.Analyses of anthocyanin of C.miniataThe anthocyanin composition and content of C.miniata the orange petals,the purple leaf and the petal three color parts(orange part,yellow part,white part)was analysed by HPLC-MS.Results show that : The Pelargonidin was identified in Clivia flowers and the Delphinidin was identified in Clivia leaf,The content was: the orange part more the yellow part more white part.2.Cloning of Chalcone Synthase and Flavanone 3-hydroxylase in Clivia miniataCmCHS and CmF3 H genes cDNA sequences were successfully obtained by degenerate primers,which were designed by NCBI databases,and RACE techniques.3.Chalcone Synthase(CmCHS)and Flavanone 3-hydroxylase(CmF3H)genes expression analysisAccording CmCHS and CmF3 H genes cDNA sequence to design specific primers.The expression pattern of CmCHS and CmF3 H genes in the main coloring organization and orange flowers at development stages were analyzed by SqRT-PCR.The results show that CmCHS and CmF3 H genes expression correlation with organization color.4.Vector constructionEukaryotic expression vector pBI121-CmCHS and pBI121-CmF3 H and prokaryotic expression vector pET28-CmCHS were successfully constructed.5.To validation gene functions by Arabidopsis mutants transformCmCHS and CmF3 H genes were transferred into Arabidopsis mutants.After transformation,T2 seeds and seedling growing point color were restored.These results demonstrated that CmCHS and CmF3 H genes related to color formation.6.To validation gene functions by enzyme activityPurified protein CmCHS was successfully obtained.Enzyme activity of CmCHS was tested with purified protein,Malonyl-CoA and p-Coumaroyl-Co A,and the reaction products were analyzed by HPLC.The results clearly demonstrated that CmCHS has a typical CHS enzymatic function.