Cloning and Expression of CHS and CHI Genes and Their Regulation on the Accumulation of Flavonoids in ’Cara Cara’ Navel Orange (Citrus Sinensis Osbeck) and ’Guoqing NO.4’ Satsuma Mandarin (Citrus Unshiu Marcow)
|School||Huazhong Agricultural University|
|Keywords||Citrus flavonoids chalcone synthase chalcone isomerase gene cloning real-time PCR relative quantification|
Flavonoids is one of the most important sources of antioxidant in citrus fruits, and study on the metabolism mechanism and expression model of key enzyme genes involved in the biosynthetic pathway is of great importance . In this work, two key genes, chalcone synthase (CHS) and chalcone isomerase (CHI), were cloned from fruits of ’Cara Cara’ navel orange (Citrus sinensis Osbeck) and ’Guoqing NO. 4’ Satsuma mandarin (Citrus unshiu Marcow). Expression of these two genes during fruit maturation and among different tissues or organs was quantified through real-time PCR technique. The content of total flavonoids and major components were also determined to research on the expression model of CHS and CHI genes and corresponding regulation mechanism on flavonoids accumulation. Major results are as follows:1. Changes in the contents of total flavonoids and major components during fruit maturation of ’Cara Cara’ navel orange and ’Guoqing NO. 4’ Satsuma mandarin. The contents of either total flavonoids or there major components, trans-chalcone, hesperidin and narirutin, in the peels were much higher than that of in the pulps at the same developmental stages. During fruit maturation, the contents of total flavonoids in the peels of both cultivars decreased gradually, however, changes in the pulps were slight and without significant difference (P < 0.05). A double-peak curve, in which the contents increased gradually to their peaks around the breaker stage (Nov. 5th) and then decreased slowly followed by a slight increase, was observed for all three components in both peels and pulps of ’Cara Cara’ navel orange. As for ’Guoqing NO. 4’ Satsuma mandarin, the contents of these three components in both peels and pulps basically showed a decreasing tendency, expect for a minor increase at the mature stage (Oct. 30th) in the pulps and a significant (P < 0.05) down-up-down course for hesperidin and narirutin around Oct. 20th in the peels.2. Cloning and sequence analysis of CHS and CHI genes. The length of cloned CHS and CHI genes were 1425 base pairs and 731 base pairs, and their accession numbers in GenBank were FJ887897 and FJ887898, respectively. Sequence analysis showed that their Open Reading Frames (ORF) were 1176 base pairs (64-1239 base pairs, code for 392 amino acids) and 669 base pairs (16-684 base pairs, code for 223 amino acids), respectively.3. Relative quantification of CHS and CHI genes during fruit maturation of ’Cara Cara’ navel orange and ’Guoqing NO. 4’ Satsuma mandarin. Expression of both genes basically down-regulated in the peels of ’Cara Cara’ navel orange with a bigger decreasing range for CHI gene, while in the pulps down-regulated right after the breaker stage, and before that a slight decrease was observed without significant difference (P < 0.05). AS for ’Guoqing NO. 4’ Satsuma mandarin, both genes also down-regulated in the peels but with a bigger decreasing range for CHS gene, instead. However, in the pulps, both genes basically up-regulated with undulatory tendencies.4. Contents of total flavonoids and relative expression of CHS and CHI genes in different tissues and organs of ’Cara Cara’ navel orange and ’Guoqing NO. 4’ Satsuma mandarin. Among the contents of total flavonoids in both cultivars, leaves occupied first place and stems came second. The minimum was in roots, in which the contents were 7.0% of that in leaves for ’Cara Cara’ navel orange and 5.3% for ’Guoqing NO. 4’ Satsuma mandarin. Expression of CHS and CHI genes between two cultivars was quite different. The minimum expression of both genes in ’Cara Cara’ navel orange was in leaves, in which the relative expression values (taken roots as the calibrator) were 0.02 for CHS gene and 0.06 for CHI gene, respectively. The maximum expression of CHS gene was in young pulps (5.52) and the second was in stems (3.12). While for CHI gene, roots (1.0) occupied first and stems (6.5) took the second, respectively. However, in ’Guoqing NO. 4’ Satsuma mandarin, the maximum expression of both genes was in leaves (7.86 for CHS and 2.39 for CHI) and stems (7.81 for CHS and 1.44 for CHI) took the second. The minimum expression was in roots (1.0) for CHS gene and in young pulps (0.10) for CHI gene, respectively.5. Regulation of CHS and CHI gene expression on flavonoid accumulation. During fruit maturation of both cultivars, expression of CHS and CHI genes was found high correlated. Their correlation coefficients were 0.90 (in peels) and 0.96 (in pulps), respectively, in ’Cara Cara’ navel orange, and were 0.98 (in peels) and 0.85 (in pulps), respectively, in ’Guoqing NO. 4’ Satsuma mandarin. High correlation was also observed between CHS or CHI gene expression and total flavonoids content. The correlation coefficients were 0.89 (CHS) and 0.81 (CHI) in peels of ’Cara Cara’ navel orange, and were 0.89 (CHS) and 0.95 (CHI) in peels of ’Guoqing NO. 4’ Satsuma mandarin, respectively. Further more, correlation coefficients between gene expression and content of trans-chalcone came up to 0.94 (CHS) and 0.99 (CHI) in peels of ’Guoqing NO. 4’ Satsuma mandarin, and high correlation was also observed between gene expression and the other two components (r = 0.62-0.78). Therefore, CHS and CHI genes together made up of the rate-limiting enzymes and involved in flavonoid regulation directly, but the regulation mechanism remained not very clear yet due to other complicated components (such as sugars, acids, carotenoids et. al) in citrus fruit.