Cloning Of The Key Genes PSY And CCS Relation To Carotene Synthesis Pathway And Plant Regeneration In Tagetes Erecta

Carotenoids in plants are indispensable auxiliary pigments and photooxidation protectants for photosynthesis process,and also constitute an important component of flower color,which has important agronomic value.Marigold(Tagetes erecta Linn)is compositae marigold annual herbaceous flower,which has become one of the most commonly used ornamental flowers in the flower bed,the flower border and other landscape gardening in China,because its colorful,long flowering,compact design,and easy to cultivation.The petals of marigold are abundant in lutein and rich in variety,which is an important model plant for researching the synthesis and accumulation of carotenoids.It is of great significance to explore the correlation between gene expression and lutein content of marigold for researching the regulation mechanism of carotenoid accumulation.The results of preliminary study show that,the expression levels of the carotenoid synthase genes PSY and CCS in the petal of marigold exist great difference in orange red inbred lines V-01 and yellow inbred lines V-01M(from V-01 mutation of orange red inbred lines)at the early colouration stage.The temporal and spatial expression patterns of PSY and CCS in marigold petals and the correlation between their expression level and color of petals have not been further studied,and function of PSY and CCS genes in marigold has not been verified.Based on this,this study cloned PSY and CCS gene,and used fluorescence quantitative PCR to analyze the temporal and spatial expression patterns in marigold petals and the correlation between their expression and color of petals.Then analyzed the effects of PSY and CCS on excessive or suppressed expression on the color of longevity chrysanthemum,and clarified the regulating effect of PSY and CCS in the synthesis of marigold petal carotene.The following main research results are as follows:1.Four genes expressed in different colors were found by analyzing gene spectrum expression of Marigold orange-red inbred line v-01 and mutant yellow inbred line v-01 m,including the key gene CCS in the synthetic pathway of the carotenoids,and the others are homologous genes PSY1,PSY2,PSY3.2.The homologous comparisons and phylogenetic tree analysis of CCS and PSY,the key genes in the synthetic pathway of marigold carotenoids cloned from other species,were performed.The results showed that The CCS gene of Te CCS is closely related to Helianthus annuus L and is the most distantly related to the CCS gene of Vitis vinifera.The PSY1 gene of Te PSY1 is closely related to Sargassun thunbergii and is the most distantly related to the PSY1 gene of Lyciun barbarum L.The PSY2 gene of Te PSY2 is closely related to Mespilus germanica and is the most distantly related to the PSY2 gene of Zoysia.Te PSY3 is closely related to Helianthus annuus L and is the most distantly related to the PSY3 gene of Lycopersicon esculentum.3.The key gene CCS and PSY in the synthesis pathway of marigold carotene were cloned.The expression level of Te CCS gene in the Marigold flowers in orange-red inbred line and mutant yellow inbred reached the lowest in the third period.The expression of the gene was highest in the second period of the orange-red inbred line,and the highest expression was in the fourth period of the yellow mutant.There was no significant change in the expression of Te PSY1 gene in the four flowering periods of marigold and yellow mutant flowers.The expression of the gene was highest in the fourth period of the marigold,and the lowest expression in the second period,the highest in the second period of the yellow mutant,and the lowest in the fourth period.The expression of Te PSY2 gene in the yellow mutant of marigold was higher than that of tangerine,and the expression level in the fourth period was the highest,and the expression quantity of the second period was the lowest.The expression of Te PSY3 gene was significantly higher in the four flowering stages of marigold yellow mutant flowers than that of the orange-red inbred line,and the expression was lowest in the first period of the two materials blooming.The expression quantity of the fourth period was the highest in the orange-red inbred line,and the second period of the yellow mutant was the highest.4.Marigold regeneration system was established,and adding different ratio of hormone concentration of 6-both BA and IAA shows impact on callus induction of marigold,and the induction rate and plant regeneration rate were the highest in 3.0mg/L6-BA and 2.0 mg/LIAA.