Transcriptome Analysis And Molecular Mechanism Of Carotenoid Synthesis Pathway Of Marigold

With the improvement of living standards,the replacement of synthetic pigments by natural pigments has gradually become a trend.Marigold(Tagetes erecta)has a large amount of pigment substances,especially carotenoids,and is an important plant resource for carotenoids extraction.Nowadays,the processes of the carotenoid synthesis pathway and the enzymes that catalyze related reactions have been well understood,but the detail functional genes and their differential expressions in this pathway of specific plants are still need to be syste matically analyzed,especially carotenoid-rich plants such as marigold.Despite the high nutritional health and economic value of marigold,the reported genetic information is very limited.In this study,eight marigold varieties were selected for planting.By comparing the yield related agronomic traits and the lutein content in flowers,the cultivar ―Juwang‖ was identified as high-quality variety resource for further experiments.Nine cDNA libraries were constructed from "Juwang" leaves,immature flowers and mature flowers for transcriptome sequencing performed by Illumina Hiseq 2000.The raw sequences were assembled and annotated.A total of 45.66 Gb nucleotides were obtained,and 72,499 unigenes were assembled.34,892(48.13%)gene sequences were annotated in NR,Swiss-prot,COG,GO,KOG,KEGG,and Pfam public databases,a large proportion of which associated with secondary metabolite biosynthesis pathways.Furthermore,the differentially expressed genes in different tissues and developmental stages of marigold were analyzed.11,721 significant differential genes were identified,which were mainly enriched in secondary metabolite biosynthesis and photosynthesis pathways.At the same time,351 stable expression genes were defined by transcriptomics information analysis,and 7 candidates were selected for expression stability evaluation as reference genes by quantitative verifiation.Using transcriptome data,the analysis of carotenoid synthesis pathway in marigold was then focused on.The genes sequence,number of transcripts,and the trend of expression changes in different tissues and flower development stages were determined.The results indicated that gene expansions of catalytic enzymes in the carotenoid synthesis pathway of marigold.It showed a possible mechanism of high pigment content in T.erecta.Moreover,using the new identified internal reference gene,the expression level of carotenoid biosynthesis pathway-related genes was detected by quantitative PCR,and four full-length sequences of genes were cloned and sequenced,which further verified the transcriptome sequencing assembly and analysis results.In addition,the marigold carotenoids synthesis genes TePDS1 and TeLCYB were introduced into tomato by Agrobacterium-mediated transformation to investigate their function and potential application in fruit pigment quality.In conclusion,this study provides theoretical basis for the molecular mechanism elucidation of plant carotenoids synthesis,and new genetic resource for high-carotenoids crop varieties cultivation and food nutritional value improvement.