| Marigold(Tagets erecta L.),a economic flower and its ray florets are high-quality plant source materials for extracting lutein,the study of the formation mechanism of its flower color difference is of great significance.In this paper,'2013-1' and '2013-2' were used as experimental materials.Using technology of high performance liquid chromatography and quantitative Real-time PCR to analyse and the composition changes of carotenoid and the expression characteristics of relative enzyme genes in carotenoid metabolism of two different color marigold varieties during four different developmental stages(S1,closed bud;S2,semi-open bud;S3,open flower;and S4,fully open flower),and obtained a gene of carotenoid cleavage dioxygenase CCD1 and CCD4 b by homology cloning,to explore the key enzyme gene of carotenoid biosynthesis and degradation and the marigold flower color formation mechanism.The results are as follows:1 Analysis of carotenoid content in marigold flowerDetecting five pigments composition of metabolic pathway of lycopene,?-carotene,?-carotene,zeaxanthin and lutein in marigold ray floerts during four different developmental stages by HPLC.The results showed:(1)Two different varieties were detected to contain zeaxanthin,?-carotene,?-carotene and lutein,and the lycopene was detected only from '2013-1',which indicated that the two varieties contained metabolic branch pathway of lutein and zeaxanthin;(2)5 kinds of carotenoid content in '2013-1' were higher or significantly higher than that of '2013-2',and the content of ?-carotene was significantly higher thanthat of the other 4 kinds of carotenoid.At the stage S3,the content of?-carotene was significantly lower than that in the other 3 stages of'2013-1',while zeaxanthin was the highest and significantly higher than other stages.Lutein was significantly lower than the other 3 stages at S1,4 stages of the flower appearance color changing may be related to the content changes of zeaxanthin,lutein and ?-carotene.The content of?-carotene in '2013-2' was significantly higher than that of the other 4kinds of carotenoid,and 4 stages presents the change tendency of 'high to low',but there was no significant difference of lutein content in the 4stages,?-carotene and zeaxanthin were detected low content,while there was no lycopene found in '2013-2'.2 Expression analysis of marigold ray floret of carotenoid biosynthetic related enzyme genesExpression analysis all carotenoid biosynthetic relative genes by Real-time PCR,results as follows:(1)The expression analysis of five genes showed that PSY,CRTISO and Z-ISO were correlated with the differences of color in two varieties,However,the expression of pale yellow '2013-2' was higher than that of the deep orange '2013-1' one.The expression of ZDS at S1 was lower or significantly lower than that of the other 3 stages in '2013-1',and there was a high positive correlation with lycopene content,while the expression of '2013-2' was highest at S4 andsignificantly higher than hat of the other 3 stages,at the same time,there were no lycopene detected out;The expression of PDS-1 was about10 times of PDS-2 in 2 varieties,the expression of PDS-2 in '2013-2' was significantly higher than '2013-1',except S1,while expression of PDS-1in '2013-1' was higher or significantly higher than '2013-2'.(2)In the zeaxanthin branch,the expression level of ZEP and NXS is high in stage S3 and S4,and the difference between the two varieties significantly,and the trend is basically consistent with flower color change,indicating that these 2 genes may be related to the two different varieties of marigold color formation.The trend of CHYB change was not obvious inthe 4 stages of the 2 varieties,but it was different from the previous reports.(3)In lutein branch,there was no significant difference between the 4stages of LCYe,But the change trend of lutein content was basically the same,and are highly significant correlation of the '2013-1' to both,speculating that LCYe may be a key enzyme in the synthesis of lutein.The expression level of CYP97 A in the 2 varieties was highest at S3,and the gene expression of was moderate or highly positively correlated with the content of lutein,speculating that CYP97 A may be a key enzyme in the synthesis of lutein.3 Expression analysis of carotenoid degradation of marigold flower related enzyme genes(1)CCD familySeven CCD genes have been found in the marigold bud transcriptome,and their expression had been detected in four different stages of two varieties by Real-time PCR.The results as follows: At S1 and S3,expression of CCD1 was the lowest in '2013-1',and at S4 was significantly higher than other periods.The relative expression of CCD1 in '2013-2' was higher than that of '2013-1' and there was a significant difference at S3;The relative expression of CCD4 a in '2013-1' is basically the same as that of flower color,but there was no significant relationship between 4 stages in '2013-2';At S3 and S4,the expression of CCD4 b were significantly higher than those of 2 stages in 2 varieties;The expression of CCD7 in the4 stages of '2013-2' was significantly higher than that of '2013-1',while NCED2,NCED3 and NCED5 are just the opposite,and it is speculated that CCD1 and CCD4 b may be the key degraded enzyme gene form 2 different varieties of marigold.(2)Other degradation related enzyme geneThe change trend of CCS in the '2013-2' is similar to that of?-carotene,but there was no significant correlation between the 4 stages in'2013-1',which showed that CCS may be related to the accumulation of in'2013-2'.4 Gene cloning analysis of CCD(1)The full-length sequence of CCD1 c DNA obtained from 2013-2marigold is 1 746 bp,which Gen Bank accession number is KX557488 with a coding region length of 1 623 bp,putatively encoding 541 amino acids;Protein analysis indicated that Te CCD1 is an unstable protein and has no signal peptide,which belongs to the RPE65 superfamily(Gen Bank accession number is PF03055)having the same conserved domain of CCD family,and it mainly located in the cytoplasm;CCD1 nucleic acid sequence of 2013-2 marigold is 89% homologous to that of Pyrethrum;Amino acid sequence analysis suggested that CCD1 of 2013-2 marigold is93% homologous to that of Pyrethrum,and 75%—83% homologous to that of 19 different species,indicating that Te CCD1 is highly conserved gene;Phylogenetic analysis showed that the evolution of Te CCD1 is basically in accordance with the evolution law of plant taxonomy and has obvious characteristics of species,which has a closest relationship with that of the species in Compositae.(2)CCD4b was homology cloned(Gen Bank accession number is KY450708).Sequence analysis showed that the full-length sequence of Te CCD4 b c DNA is 1 725 bp,with a coding region length of 1 392 bp,putatively encoding 463 amino acids;The encoded protein Te CCD4 b was mainly localized in the plasma membrane,which belongs to the RPE65 superfamily having the same conserved domain of CCD family;Homology analysis indicated that high homology between Te CCD4 b,Ha CCD4-L of Helianthus annuus and Cm CCD4 b of Chrysanthemum × morifolium;Phylogenetic analysis suggested that the close relationship between Te CCD4 b,Ha CCD4-L and Cm CCD4 b,and were different from CCD4 and CCD4 a of other species,which showed that Te CCD4 b is not very conservative in the process of evolution,and there were significant differences among different species. |
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