论文标题:茶树类胡萝卜素累积及其相关基因表达模式研究
Carotenoids Accumulation and Expression Pattern of Carotenogenic Genes in Tea [Camellia Sinensis (L.) O.Kuntze.]
论文作者
论文导师 梁月荣,论文学位 博士,论文专业 茶学
论文单位 浙江大学,点击次数 399,论文页数 112页File Size7655K
2008-04-01论文网 http://www.lw23.com/lunwen_2516627/
Carotenoids;;β-carotene;; Xanthophyll;; carotenogenic genes;; phytoene synthase;; phytoene desaturase;; violoxanthin deepoxidase;; albino mutant;; RACE;; phylogenetic tree
茶[Camellia sinensis(L.)O.Kuntze]是一种用于生产无酒精饮料的多年生木本植物,被称为传统的“亚洲饮料”,在茶叶生产国的经济具有重要地位。随着全球茶叶生产国及其产量的增加,国际竞争加剧,生产国的研究精力更多地由原来重视产量转向重视质量。茶叶质量是一个复杂的因素,其中香气化合物含量和组成是影响成品茶叶感官品质的重要决定因子。有研究报道,茶叶中的许多香气化合物都来源于类胡罗卜素。然而,相关的文献报道却不多。深入了解不同茶类,如红茶、绿茶和乌龙茶栽培品种的类胡罗卜素含量、在不同收获季节的含量变化及其与所生产茶类及香气形成的关系,对于进一步掌握类胡罗卜素与茶叶感官品质之间的关系有重要意义,而且十分必要。 类胡罗卜素不但对植物来讲具有一系列的特殊功能,而且也是动物和人类的膳食中也是重要的成分。在过去的若干年份里,几乎所有与编码绿色植物类胡罗卜素生物合成相关酶类的基因和cDNA都已经进行了鉴定或测序,有关编码的蛋白质等产物的特性也进行了分析。综合有关类胡罗卜素生物合成相关基因的信息,了解这些基因表达在类胡罗卜素生物合成及累积中的作用,对于开发调节类胡罗卜素代谢途径,改变类胡罗卜素的累积方式等方面都具有重要意义。 本研究选择中国生产红茶、绿茶和乌龙茶代表性品种12个,研究其在不同采收季节种类胡罗卜素累积的情况。八氢番茄红素合成酶(phytoenesynthase,PSY)和八氢番茄红素脱氢酶(phytoene desaturase,PDS)是类胡罗卜素生物合成途径中第一步和第二步的催化酶。堇菜黄质去环氧酶(violaxanthin de-epoxidase,VDE)是类胡罗卜素生物合成途径中叶黄素循环的关键酶。本文鉴定了茶树中的该3种酶基因(Accession no:PSY,EF545005;PDS,EU275984 and VDE,EU091350),研究了不同收获季节这些基因的表达模式,同时对低温敏感型叶绿素缺乏的白化变异体茶树中这些酶基因表达进行了比较。 研究结果显示,供试的12个品种在不同采摘季节的类胡罗卜素含量差异显著,其变化幅度甚至大于品种之间的变异。就品种之间比较而言,绿茶品种龙井-43的类胡罗卜素含量最高;但从同一季节的前后期比较,后期收获的鲜叶类胡罗卜素含量高于前期收获的鲜叶。以香气品质突出的乌龙茶,一般是利用每个季节后期的成熟新梢加工而成。这也意味着类胡罗卜素含量在茶叶香气形成过程中具有重要作用。 为了分析类胡罗卜素以及多酚类含量与挥发性香气化合物(volatile flavourcompound,VFC)之间的关系,本研究对12个供试品种的春梢早期的鲜叶固定样品进行了检测。结果表明,类胡罗卜素与VFC之间不存在简单的线性关系。对于类胡罗卜素起源的VFC需要准确的进一步定性和定量鉴定以及对类胡罗卜素降解酶类活力的深入研究,才有可能了解类胡罗卜素与VFC形成之间的内在关系。虽然有研究表明,高含量多酚类对VFC的形成是有阻碍作用的,但本研究结果发现,多酚类含量与VFC之间也不存在直接的统计学关系。 用RT-PCR半定量方法分析了绿茶品种龙井-43和红茶品种玉兰的PSY,PDS和VDE基因表达谱,同时测定了相应季节的类胡罗卜素含量。研究揭示,PSY和PDS基因表达量与类胡罗卜素类及量之间存在明显的相关关系。但PDS基因的表达量远远低于PYS基因,某些季节甚至不表达。VDE酶的作用是催化堇菜黄质(violaxanthin)转化为玉米黄质(zeaxanthin),而玉米黄质是一种具有光保护功能的化合物。在逆境条件下VDE基因具有较高的表达。本研究结果证明,在高温强光的夏季,VDE基因大量表达。但VDE基因的表达与堇菜黄质含量之间不存在相关性。 在低温诱导的白化变异体茶树中,白化叶片的PSY,PDS和VDE基因表达高于同植株的绿色叶片。进一步的分析发现,白化叶片中类胡罗卜素含量很低,叶绿体发生岐变。类胡罗卜素生物合成途径相关酶的基因是核基因,但也参与质体的生物合成途径。根据质体信号传导学说理论,编码质体酶的核基因的表达受到质体信号的调节。因为在具有岐变质体组织中发现PSY,PDS和VDE的高表达,说明这些基因的表达是受质体信号的负向调节的。 PSY基因表达与茶叶类胡罗卜素的累积呈现正相关关系,本研究应用cDNA末端快速扩增的3"-RACE和5"-RACE技术克隆了PSY全长基因序列。根据推导的氨基酸序列建立了茶树PSY基因与其他作物种PSY基因系统发生进化树。结果显示,茶树PSY基因与Adonis palaestina和Citrus sinensis的关系最密切,相似性均达到90%。 总之,本研究揭示了类胡罗卜素是茶叶的重要化学成分,它不但具有与其它作物同样的生理和营养功能,还参与茶叶香气成分的形成,在决定茶叶品质方面有重要作用。PSY基因表达量高,而且与茶叶类胡罗卜素累积量具有正相关关系。我们认为,PSY,基因可以作为鉴别茶树品种类胡罗卜素合成潜力的分子标记,应用于茶树育种的早期鉴定中。
Tea [Camellia sinensis (L.) O. Kuntze] is a perennial crop and widely cultivated for the production of a popular non-alcoholic beverage. Known as the traditional "Asian beverage", tea plays a significant role in the economy of the producer countries. With the increased participation of producer countries in the global tea market, research efforts in recent years, diverted from quantity to quality. Quality of tea is a complex character and the volatile flavor compounds in the finished product are strong determinant of the quality. Many of the flavor compounds reported from tea were of carotenoids origin, however, literature on this aspect in tea is sporadic. Understanding of the carotenoids content in different tea cultivars used for production of black, green and oolong tea, their fluctuation in different harvesting seasons and relation to different kinds of tea are required for understanding the relation of carotenoids content to tea quality. Carotenoids, along with their extraordinary variety of functions in plants, are important component of animal diets, including human. In the past few years, genes and cDNAs encoding nearly all the enzymes required for carotenoids biosynthesis in green plants have been identified and sequenced and their products characterized. Identification and sequencing of the carotenogenic genes are required to gather information how the expression of carotenogenic genes regulates the accumulation of the carotenoid and to manipulate the caretenoids pathway for enhancing the carotenoids accumulation. In the present investigation we studied the carotenoids accumulation pattern in twelve tea cultivars representing elite tea cultivars of black, green and oolong tea in different harvesting seasons. Phytoene synthase (PSY) and phytoene desaturase (PDS) are the two regulatory enzymes in the carotenoids pathway catalyzing the first two steps of the pathway. Violaxanthin deepoxidase (VDE) is another key enzyme in the xanthophylls cycle within the carotenoids pathways. We identified these three genes in tea (Accession no: PSY, EF545005; PDS, EU275984 and VDE, EU091350) and studied the expression pattern of these genes in different harvesting season. The expressions of these genes were also studied in the white and green tissues of a thermo-sensitive chlorophyll deficient albino mutant of tea. A significant variation was found for the carotenoids contents in all the studied cultivars and in the plucking seasons. The variation was more pronounced between seasons than between cultivars. Longjing-43, a popular green tea cultivar in China, accumulated the highest carotenoids. In every season, high accumulation of the carotenoids was detected in the later period of the season than the early period. Since flavoury oolong tea is produced from shoot plucked in the late of the season, it implies the role of carotenoids in the flavour formation in tea. To establish a relation of the flavour compounds with that of the carotenoids content, we estimated the volatile flavour compounds (VFC) in the steam fixed material of the twelve cultivars plucked in the early spring season. However, our investigation could not establish a stable relation of VFC to the carotenoids content. More systematic study, particularly accurate identification of VFC of carotenoids origin and their modification, determination of the quantity and activity of carotenoids cleavage enzymes will provide more insights into the role of carotenoids on flavour formation. Polyphenol content was reported to hinder the formation of VFC. We determined the polyphenol content in the material used to estimate the VFC content. However, our investigation could not establish such a relation. The expression of the PSY, PDS and VDE were studied in two cultivars: Longjing-43, a green tea cultivar with small leaf and Yulan, a black tea cultivar with large leaf. The expression of these genes in the early and late period of the three plucking seasons were measured by a semi-quantitative RT-PCR method and the carotenoids content were estimated in the respective seasons. The study revealed a strong relation of PSY and PDS expression to that of carotenoids accumulation. However, the transcript abundance of PDS is much more low than that of PSY and this gene even failed to amplify in some seasons. VDE catalyzes the conversion of Violaxanthin to zeaxanthin, a compound having photo-protective function and high expression of this gene was reported in stress conditions. As expected, in our investigation, we observed a higher expression of this gene in sunny summer periods. However, expression of this gene did not show a high relation to the Violaxanthin content. The expression analysis of the PSY, PDS and VDE in the white tissue of the albino mutant revealed a high expression in comparison with that in its green counterpart. The white tissue was found to accumulate low carotenoids and have damaged chloroplast. The carotenogenic genes are nuclear encoded but participate in the biosynthetic pathway in the plastid. According to the plastid signal hypotheses, the expression of the nuclear encoded plastid enzymes is regulated by the signal from the plastid. Since high expression of these genes were observed in the tissue with damaged plastids, expression of these genes may be regulated by negative plastidic signal. The expression of the PSY gene was found to positively correlated with the carotenoids accumulation in tea and the full length PSY gene was obtained by performing 3"- and 5"- RACE. The evolutionary relationship of the deduced amino acid sequence of the obtained PSY from tea with that reported from other crop species was inferred by constructing a phylogenetic tree. Result showed that PSY in tea was closely related with that of Adonis palaestina (90%) and of Citrus sinensis (90%). It can be concluded from this investigation that carotenoids content, as reported in other plant system, is also an important chemical component in tea and plays a significant role in determining tea quality. Since the PSY gene can be easily amplified and its expression showed a positive correlation with the carotenoids accumulation, this gene can be a suitable candidate for designing a molecular marker for carotenoids content.
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