Cloning And Functional Analysis Of Carotenoid Biosynthesis Pathway Genes In Tagetes Erecta

Plant secondary metabolic terpenoids show structural diversity due to their different condensations and derivative modifications and thus biological activity diversity,especially in plant-environment interaction.Among them,carotenoids,one of tetra terpenoids,and chlorophyll,with diterpene side chain,play irreplaceable roles in photosynthesis and antioxidation.They are also main pigments of plants,their synthesis and accumulation to various contents in different plant tissues can induce animal behaviors in pollination and transmission,and also endow plants with horticultural ornamental values.As an ornamental plant,marigold(Tagetes erecta L.)is widely planted all over the world because of its gorgeous flower color.Moreover,it has become an important resource for natural pigments extraction due to the rich in carotenoids.The high pigment content implies the high efficiency of the genetic genes in carotenoid synthesis pathway of marigold.Therefore,in this study,the carotenoid biosynthesis related genes(Te PSY,Te ZDS,Te CHY,Te CYP,Te CCS,Te CCD4,Te NCED2)and chlorophyll synthesis gene(Te PPO)were cloned from high pigment marigold variety by using the transcriptomic data.The full-length coding sequences of the eight genes were obtained by sequencing validation.Furtherly,phylogenetic evolution trees were constructed with homology modeling method to analyze the evolutionary relationship of these genes among plant species,and the results showed the highest similarity with sunflower homologs submitted in the database.Additionally,physical-chemical properties analysis and three-dimensional structure prediction of the encoded protein also provide bioinformatic clues for their functions.Moreover,the plant expression vectors of Te ZDS,Te CHY,Te CYP,Te CCS and Te NCED2,putative carotenoid catalytic enzymes in Tagetes erecta,were constructed for further functional analysis.Te ZDS,encoding ζ-carotene desaturase to catalyze the synthesis of lycopene in the upstream of the pathway,was transformed into the fruit model plant of tomato.The tomato lines expressing Te ZDS were obtained.The phenotypic analysis indicated that there were no significant differences in plant height,plant or leaf shape,and flowering time between transgenic plants and wild type,but the tomato fruits transferred with Te ZDS turned red early and showed deeper color at maturity.The pigment determination displayed significantly higher contents of lycopene and β-carotene in Te ZDS transformed tomatoes,which suggesting that the introduction of marigold Te ZDS can improve the pigment quality without significant adverse effect on the growth and development of tomato plants.The results indicated that Te ZDS has functional activity for carotenoid synthesis and application potential for genetic improvement on pigment nutrition in fruit crops.In addition,the quantitative expression analysis of Te PPO in different color marigold flowers at different developmental stages suggested that chlorophyll synthesis related gene Te PPO may be involved in green coloration of marigold.In summary,eight marigold genes of carotenoid synthesis and flower color formation were cloned in the present work and Te ZDS was transformed into tomato for function analysis,which provide new gene resources and research basis for exploring the molecular mechanism of carotenoid accumulation in high pigment plants and thus improvement of pigment nutritional quality in fruit crops.