The Function Of The Lycopene ?-Cyclase Gene From Chlorella Vulgaris

LCYE(lycopene ?-cyclase)is a key enzyme in the carotenoid evolution pathway,which can catalyze the cyclization of lycopene,in turn affects the synthesis of lutein.Lutein is a kind of carotenoid with highly value.It is natural colorant that has been widely used in the food industry.Because of its high antioxidant activity,it also has a great impact on human health.Extraction from marigold is its main source,but efficiency is limited by many factors,therefore,lutein demand is still a problem to be solved.CvLCYE is the lycopene ?-cyclase gene of Chlorella vulgaris F197.We amplified and cloned to obtain the full length of the gene for the first time,but the structure and enzyme activity of this gene have no direct data so far;therefore,the gene structure was analyzed;then the enzyme activity was compared in vitro to explore the enzyme activity of CvLCYE;then gene CvLCYE was transformed into Chlorella vulgaris and Chlamydomonas reinhardtii respectively to explore the gene function in vivo;finally,C.reinhardtii and C.vulgaris with high yield of lutein were screened.The specific conclusions obtained from the experiment were as follows:Gene structural analysis and the subcellular localization of CvLCYE.We designed a degenerate primer by cross-contrast the conserved regions of LCYE from Chlorella variabilis,Auexnochlorella protothecoides and Chlamydomonas reinhardtii,and amplified and obtained CDS fragments of CvLCYE then isolated the full-length gene of CvlcyE by RACE.Nucleic acid sequence and amino acid of CvLCYE,was compared with CvalcyE from Chlorella variabilis,ApLCYE from C.protothecoides,CrLCYE from C.reinhardtii,and CzLCYE from Chlorella zofingiensis.The results show that CvLCYE has the highest homology with ApLCYE and CvaLCYE,then the CzLCYE,and the homology with CrLCYE is the weakest.To explore the subcellular localization of the gene,we constructed pUC19-gfp-CvLCYE vector,transformed it into the protoplasts of Arabidopsis,and determine the specific location of CvLCYE in the cell using the luminescent position of GFP.Finally we found: most of CvLCYE is located in the chloroplast,and a small part is located in the cell membrane.To compare the enzymatic activities of LCYE from four microalgae,we constructed CvLCYE,ApLCYE,CzLCYE and CrLCYE into pUC19 vector respectively,and obtained recombinant plasmids containing different LCYE.The recombinant plasmids and plasmid pACCRT-EIB which can accumulate lycopene in bacteria,were co-transformed into E.coli to obtain recombinant.The content of lycopene in the recombinant E.coli cells was detected by HPLC,and the enzyme activity of LCYE was compared by the content of lycopene.Finally,it was concluded that ApLCYE had the strongest activity;CvLCYE was the second.However,CvLCYE has little difference compared with ApLCYE;CzLCYE has relatively low enzyme activity;while CrLCYE has the weakest enzyme activity.To explore the function of CvLCYE in C.vulgaris,we constructed recombinant plasmid of pCAMBIA-CvLCYE,and pOX-CvLCYE,and transformed them into Agrobacterium tumefaciens.With the infectivity of Agrobacterium,CvLCYE was introduced into the genome of chlorella,and the recombinant chlorella that overexpressed CvLCYE was obtained.The content of lutein was detected by HPLC.The results showed that the recombinant Chlorella had higher lutein content than the wild type Chlorella,and the highest was 1.81 times as high as the wild-type.To explore the function of CvLCYE in C.reinhardtii,we constructed a recombinant plasmid of pH124-CvLCYE,and transformed it into C.reinhardtii strain CC124 by bead milling,and obtained recombinant C.reinhardtii that overexpressed CvLCYE.The content of lutein was detected by HPLC.The results showed that tstrain of C.reinhardti had higher lutein than the wild type strain,and the highest was 2.32 times of the wild type.Based on the above experimental results,CvLCYE is highly homologous to ApLCYE and CzLCYE,and plays a similar role.CvLCYE has strong enzymatic activity in vitro,and can increase lutein content in both C.vulgaris and C.reinhardtii.This provides a basis for better understanding the functions of CvlcyE and a new pathway for biosynthesis of lutein.