【作者】 李静；

【导师】 夏仁学； 易干军； 李国怀； 彭抒昂； 徐娟； 程运江；

【作者基本信息】 华中农业大学 ， 果树学， 2009， 硕士

【摘要】 类黄酮(Flavonoids)是柑橘果实中抗氧化活性成分的重要来源之一,研究其代谢机制及其合成途径中关键酶基因的表达模式对柑橘类黄酮的开发和利用至关重要。本文以红肉脐橙(Citrus sinensis cv.Cara cara)和‘国庆四号’温州蜜柑(Citrusreticulata Blanco cv.Guoqing NO.4)为试材,克隆类黄酮合成途径中的两个关键酶基因——查尔酮合成酶(CHS)和查尔酮异构酶(CHI),应用real-time PCR技术检测其在果实成熟过程中及不同器官和组织中的相对表达量,结合总黄酮含量及主要类黄酮组分的变化,探讨了CHS和CHI基因的表达模式及其对类黄酮积累的调控机制。主要研究结果如下:1、红肉脐橙和‘国庆四号’温州蜜柑果实成熟过程中总黄酮及三种主要类黄酮组分含量的变化规律。无论是总黄酮还是反式查尔酮、橙皮苷和柚皮素-7-β-芸香糖苷,它们在两品种果皮中的含量均远高于其在同时期果肉中的含量。随着果实的成熟,总黄酮在两品种果皮中的含量均逐渐下降,而在果肉中变幅较小且差异不显著(P＜0.05)。三种类黄酮组分在红肉脐橙果皮和果肉中的含量变化基本一致,均呈双峰变化趋势,即三者含量均先逐渐上升至转色期(11月5日)前后达到其峰值而后逐渐下降,最后又有所升高。随着果实的成熟,三种类黄酮组分在‘国庆四号’温州蜜柑果皮和果肉中的含量均基本呈下降趋势,但在果肉完全成熟时(10月30日)又均有所升高,而果皮中橙皮苷和柚皮素-7-β-芸香糖苷的含量在转色之后的10月20日前后都有一个先显著下降、然后显著升高、再下降的过程(P＜0.05)。2、查尔酮合成酶(CHS)和查尔酮异构酶(CHI)基因克隆及其序列分析。克隆的CHS和CHI基因碱基序列长度分别为1425 bp和731 bp,在GenBank中的登录号分别为FJ887897和FJ887898。序列分析结果显示,其开放读码框(ORF)分别为1176 bp(64～1239 bp,编码392个氨基酸)和669 bp(16～684 bp,编码223个氨基酸)。3、红肉脐橙和‘国庆四号’温州蜜柑果实成熟过程中CHS和CHI基因的相对表达量。CHS和CHI基因在红肉脐橙果皮中均基本呈下调表达趋势,但CHI基因的下调幅度较大;在果肉中均是转色期(11月5日)之后下调表达,之前略有下降但变化不显著(P＜0.05)。两基因在‘国庆四号’温州蜜柑果皮中也均为下调表达,但CHS基因的下调幅度较大;两者在果肉中均基本呈上调表达趋势,但表达量起伏波动较大。4、红肉脐橙和‘国庆四号’温州蜜柑不同器官和组织中的总黄酮含量及CHS和CHI基因的相对表达量。两品种总黄酮含量均是叶中最高、茎次之,根中含量最低,仅分别为其叶中总黄酮含量的7.0%和5.3%。CHS和CHI基因表达在两品种间的差异较大,两基因在红肉脐橙中均是叶中的相对表达量最低,分别为0.02和0.06(均以根中表达量为参照值),其中CHS基因在幼果果肉中的表达量最高(5.52)、茎次之(3.12),CHI基因则是根中最高(1.0)、茎次之(0.65)。两基因在‘国庆四号’温州蜜柑中的相对表达量则均是叶中最高(分别为7.86和2.39)、茎次之(分别为7.81和1.44),CHS和CHI基因在根(1.0)和幼果果肉(0.10)中的相对表达量最低。5、CHS和CHI基因表达对类黄酮积累的调控机制。两品种果实成熟过程中,CHS与CHI基因表达高度相关,其相关系数在红肉脐橙中分别为0.90(果皮中)和0.96(果肉中),在‘国庆四号’温州蜜柑中分别为0.98(果皮中)和0.85(果肉中)。CHS、CHI基因表达与总黄酮含量也高度相关,在红肉脐橙果皮中分别为0.89(CHS)和0.81(CHI),在‘国庆四号’温州蜜柑果皮中分别为0.89(CHS)和0.95(CHI)。此外,两基因表达与‘国庆四号’温州蜜柑果皮中反式查尔酮含量变化的相关系数分别达到了0.94(CHS)和0.99(CHI),与另外两种类黄酮组分的相关性也均较高(r=0.62～0.78)。由此表明,CHS和CHI基因共同构成了柑橘类黄酮合成途径中的限速酶,直接参与类黄酮积累的调控,但由于柑橘果实中其它物质的成分也比较复杂(如糖、酸、类胡萝卜素等),两基因对其类黄酮积累的调控机制还不甚明朗。更多还原

【Abstract】 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.更多还原