| In parallel with the development of economy and the health demand from people daily life, the fresh fruit market requires higher fruit quality, particularly functional fruit cultivars are widely welcomed. This requires citrus breeding programs to innovate highly featured citrus varieties, or varieties rich in secondary metabolites to improve market competitiveness. Fruit color has important influence on sensory and nutritional quality of fruit, so it is an important factor influencing both the external and internal quality of fruit. Citrus color mutants have high accumulation of the antioxidant carotenoids, this make them high nutrition and health values, and larger market potential.As the biggest production and plantation area of pummelo cultivar in China, Guanxi pummelo (Citrus grandis cv. Guanxi) is the favorite pummelo of the consumer due to low sugar, juicy, rich in vitamin C and a number of other nutrient elements. Because of specific accumulation of carotenoids, the Guanxi pummelo fruit color mutants of red and yellow flesh found in recent years attract attentions from both industry and research. The fruit colors of mutants make them more appealing to consumers in the fruit market and remarkably high price. At the same time, the color mutants of natural somatic cells have important value for studying the mechanism of carotenoid accumulation in citrus. In this study, to explore the molecular mechanism of color mutants, the carotenoid species and concentration were determined on Guanxi pummelo and its yellow flesh and red-flesh mutants in different developmental stages and tissues, the carotenogenic gene expression analysis were subsequently performed. The main results are as follows:1. The carotenoid species and concentration of wild type pummelo and color mutants in four layers of tissue and five developmental stages were determined by HPLC. Carotenoid accumulation pattern shows dynamic changes in different varieties, with some tissue specificity. Red-fleshed mutant accumulates high content lycopene in mature segment membrane and juice sacs, and yellow-fleshed mutant accumulates high levels of β-carotene. That is why the two mutants show bright red and yellow. Red mutant observably accumulates lycopene in the 120 days after flowering, and the β-carotene of yellow-fleshed mutant maintains a high level during the fruit developmental process.2. The expression characteristics of carotenoid biosynthetic genes of wild type and mutants were performed by quantitative RT-PCR analysis. According to data of 13 carotenogenic genes and 3 methylerythritol phosphate (MEP) pathways, the expression of LCYe (lycopene e-cyclase) and LCYb (lycopene β-cyclase) genes decrease rapidly in the juice sacs of red-fleshed mutant during the developmental process, and in the segment membrane of red-fleshed mutant, the expressions of DXS and DXR are higher than the white and yellow. These datas suggest that the decreasing expression of related downstream genes (mainly LCYe and LCYb) may be the molecular basis of lycopene accumulation in the juice sacs of red-fleshed mutant. But there may be a different reason why segment membrane of red-fished mutant has a high level of lycopene accumulation. In addition to, the expressions of BCH and ZEP in the yellow-fleshed mutant, the key genes in downstream of carotenoids metabolic pathway, decrease rapidly from the young fruit period (60DAF, May) in the yellow-fleshed. This may be associated with the accumulation of β-carotene in yellow-fleshed mutant.3. cDNA sequences of carotenogenic genes (the rate-limiting enzyme PSY, cyclase LCYe and LCYb) from the wild type and mutants were cloned and compared. Through comparing the PSY gene sequences of Guanxi pummelo with satsuma orange (C. unshiu), grapefruit (C. paradisi) and orange (C. sinensis) included in NCBI, there are highly consistent sequences after three, but the pummelo exists multiple mutant sites of bases basis and a continuous bases sequence (AATAAT); the wild type of Guanxi pummelo and mutants are highly consistent, there are not SNP sites and continuous bases mutant. Comparing LCYB2 gene sequences of pummelo, grapefruit (C. paradisi) and orange (C. sinensis), the sequences are highly consistent of satsuma orange, pummelo and grapefruit; comparing LCYB2 gene sequences cloned from white pummelo and mutants, there are differences of individual bases (position) in yellow, T → A in the 2337 position, C → G in the 2546 position, T→ C in the 2640 position, respectively. Comparing LCYE gene sequences of white pummelo, grapefruit and satsuma orange, finding there is a missing of 14 consecutive nucleotides (TGTGTCGAATCAGG) in satsuma orange; comparing LCYE sequences cloned from white pummelo and mutants, there exist differences of individual bases in yellow(G→A in the 995 position) and red(T missing in the 1146 position). Sequence differences of color mutants in LCYB2 and LCYE genes provide foundation for future gene function and hereditary association analysis study. |
