| Marigold,a economic flower and its high-quality plant source materials for extracting carotenoid,the study of the Carotenoid metabolic pathway is of great significance.In this paper,four organs(root,stem,leaf and flower)of ’Sc’ and ’MA’ were used as experimental materials.The expression characteristics of biosynthesis and degradation related enzyme genes were analyzed by Real-time PCR,obtained genes of Cytochrome P450-type monooxygenase 97A(CYP97A)and 9-cis-epoxycarotenoid dioxygenases NCED2 by homology cloning.At the same time,the vector was constructed,transgenic to Arabidopsis thaliana,and the changes of carotenoid content in transgenic and non-transgenic Arabidopsis thaliana were detected by High Performance Liquid Chromatog raphy.To study the functional characteristics of key enzyme genes in carotenoid metabolism of marigold carotenoid.The contents of the study are as follows:1.Expression analysis of marigold carotenoid biosynthetic important enzyme genesReal-time PCR was employed to investigate the relationship between the expression of carotenoid biosynthetic related genes and the flower color of Tagetes erecta L.in two varieties and four different organ genes.The conclusions are as follows:(1)ZDS,LCYb,LCTe and CYP97 A has different expression level in roots,stems,leaves and flowers of the two marigold varieties.The expression of these four genes in the flower of ‘Sc’ was higher than that of ‘MA’(P<0.01),which suggests that all these genes may involve in the regulation of flower color of these two varieties.(2)There was no significant difference in the expression of CHYB between two marigold cultivars.It was suggested that CHYB might not be the key enzyme gene for flower color formation of marigold.2.Expression analysis of marigold carotenoid degradation important enzyme genesReal-time PCR was employed to investigate the relationship between the expression of carotenoid degradation related genes and the flower color of marigold.The results showed that CCD1,CCD4 b,CCD7 and NCED2 has different expression level in roots,stems,leaves and flowers of the two marigold varieties.The expression of these four genes in the flower of ‘MA’ was higher than that of ‘Sc’(P<0.01),which suggests that all these genes may involve in the regulation of flower color of these two cultivars.3.Cloning and functional analysis of(CYP97A)P450 cytochrome monooxygenase 97 A gene in marigoldThe full-length sequence of CYP97 A cDNA obtained from ‘Sc’ marigold is 2 184 bp,which GenBank accession number is MK061379 with a coding region length of 1 821 bp,putatively encoding 606 amino acids.Protein analysis calculate indicated that TeCYP97 A is a hydrophilic protein protein.It mainly located in the chloroplast.Homologous protein sequence alignment analysis shows that TeCYP97 A is a highly conserved gene in marigold.Phylogenetic tree studies show that TeCYP97 A belong to cytochrome P450-type monooxygenase 97 A.HPLC analysis showed that CYP97 A gene increased the contents of lutein,α-carotene,β-carotene and zeaxanthin in transgenic Arabidopsis thaliana leaves.CYP97 A may be a key enzyme gene in the pathway of carotenoid biosynthesis.4.Cloning and functional analysis of a 9-cis-epoxy-carboxycarotene dioxygenase gene(NCED2)from marigoldThe full-length sequence of NCED2 cDNA obtained from ‘MA’ marigold is 2 258 bp,which GenBank accession number is MK061380 with a coding region length of 1 791 bp,putatively encoding 596 amino acids.Protein analysis calculate indicated that TeNCED2 is a hydrophilic protein,which mainly located in the chloroplast.Protein multiple sequence alignment analysis showed that TeNCED2 had the conserved domain characteristics of NCED family,and phylogenetic tree analysis showed that TeNCED2 belonged to NECD family and protein sequence was conserved.HPLC analysis showed that NCED2 gene reduced the contents of lutein,α-carotene,β-carotene and zeaxanthin in transgenic Arabidopsis thaliana leaves.NCED2 may be a key enzyme gene in the pathway of carotenoid degradation metabolism. |
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