【作者】 郑洁红；

【导师】 邱栋梁； 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2012， 博士

【摘要】 龙眼（Dimocarpus longan Lour.）原产于我国的亚热带水果，是福建省重要的特色果树。具文献记载，龙眼含有丰富的黄酮类物质，具有很高的药用价值。前人对龙眼黄酮类物质的研究主要集中在果实中这类物质的成分与含量的研究，但对其合成机理不甚了解。因此，本研究通过比较不同品种间芦丁和槲皮素的含量差异，克隆芦丁和槲皮素合成过程中相关基因的cDNA序列和启动子序列，研究这些基因在不同品种中的表达与调控，从而明确了黄酮类物质合成途径的关键基因，为进一步揭示黄酮类物质合成途径的分子机制奠定了良好基础。取得的主要研究结果如下：1、利用HPLC技术对龙眼4个品种‘松风本’、‘石硖’、‘立冬本’和‘乌龙岭’叶片的芦丁和槲皮素含量进行测定。分析结果表明，4个品种间芦丁的相对含量具有显著差异，含量最高的是‘乌龙岭’，达到439.8μg/g，含量最低的是‘石硖’，低于260μg/g；4个品种的黄酮含量有差异显著。在对4个龙眼品种中的槲皮素测定结果表明，四个品种中，松风本含量最低，为69.6μg/g，而石硖和立冬本的相对含量差异不显著，分别为131.8μg/g和129.1μg/g，但在乌龙岭叶片中没有检测到槲皮素。2、本研究利用RACE技术，以龙眼叶片cDNA为模板克隆得到Dlpal和Dlf3h的cDNA全长序列，得到FLS的保守区序列Dlfls，生物信息学分析结果表明这些序列分别与已克隆的PAL、F3H和FLS cDNA序列具有较高的同源性，通过对Dlpal和Dlf3h的编码产物进行预测，结果也表明两者的编码产物分布具有PAL和F3H的保守结构域，初步能断定Dlpal、Dlf3h和Dlfls是龙眼叶片的结构基因，参与了黄酮类物质的合成过程。根据Dlpal cDNA序列预测龙眼Dlpal酶蛋白的分子量约为306.4KD，是个碱性的亲水蛋白，属于芳香族氨基酸解氨酶，含有PAL（PLN02457）和PAL-HAL保守结构域，与PAL和HAL具有相同的功能。通过亚细胞定位分析，推测Dlpal为N端朝内由外到内的跨膜结构，在内质网上合成，并催化苯丙素类化合物合成。根据Dlf3h cDNA序列预测Dlf3h酶蛋白分子量约为45.3KD，是个亲水性蛋白，定位于内质网膜，具有双向跨膜结构。Dlf3h的整条肽链横跨于内质网膜内外，催化5,7,4’-黄烷酮，生成2R,3R-黄烷酮醇。3、本研究采用Takara公司的TAIL-PCR试剂盒和I-PCR技术克隆了龙眼叶片中CHI、CHS、PAL、F3H基因的5’端上游序列，PlantCare分析表明所获得的CHI-P、CHS-P、PAL-P、F3H-P均含有CHS、CHI、PAL、F3H基因启动子序列。本研究所获得的5个基因的启动子序列中，都包含光反应元件，如SP1、Box4、L-box等，说明参与黄酮类物质合成的基因启动子中潜在着复杂的光应激表达模式，PAL、CHI、CHS、F3H的表达具有光依赖性。除共同含有光反应元件外，还有一些与植物激素有关的元件。根据F3H、CHS和CHI的启动子序列中都包含的脱落酸响应的顺式作用元件推断F3H、CHS和CHI在龙眼叶片的类黄酮合成过程中协同表达，受到内源激素的调节。CHI-P含有赤霉素响应的元件TCCACCT-motif，CHS-P和PAL-P含有参与茉莉酸甲酯响应的元件CGTCA-motif，CHS-P中还含有水杨酸响应的元件TCA-element。4、龙眼叶片的Dlpal、CHI、CHS、Dlf3h、Dlfls在4个品种中表现出不同的表达模式，同一个基因在不同品种中的表达量具有显著性差异，结合第二章的结果，表明在同一个品种中，这些基因的表达量与黄酮类物质芦丁和槲皮素的含量具有正相关性。因此，可以认为Dlpal、CHI、CHS、Dlf3h、Dlfls在芦丁和槲皮素的合成过程中都起到了关键作用，这些基因的转录水平决定了龙眼叶片中黄酮类物质的含量。更多还原

【Abstract】 Longan (Dimocarpus longan Lour.) is a kind of famous subtropics fruit originedfrom south of China. It is also an important fruit in Fujian province. According toliteratures, longan has abundant flavonoids in fruits and leaves, and it is considered asa medical resource. Studies on flavonoid in longan mainly focused on componentsand contents in fruits. The mechanism of flavonoid biosynthesis in longan was still inpuzzled. To confirmed key genes in flavonoid biosysthesis pathway in longan leaves,rutin and quercetin contents in4cultivar varieties were determined, some relatedgenes such as Dlpal, Dlf3h and Dlfls cDNA were cloned, the promoter sequences ofDlpal, Dlf3h, Dlfls, CHI and CHS were also obtained, and these genes’ expressionlevels in longan leaves were studied in this article. The mainly results as follows:1) Rutin and quercetin contents were measured in4cultivar varieties‘Songfengben’,‘Shixia’,‘Lidongben’ and ‘Wulongling’ by HPLC. The resultsshowed that there was significant difference in rutin contents among4varieties. Therutin content in ‘Wulongling’ is highest in4varieties with439.8μg/g. The quercetincontent in ‘Songfengben’ is lowest in4varieties with69.6μg/g. There was nosignificant difference between ‘Shixia’ and ‘Lidongben’ with the content of131.8μg/gand129.1μg/g respectively. There was no quercetin detected in ‘Wulongling’.2) Full length cDNA of Dlpal and Dlf3h were cloned by RACE technology, andpartial cDNA of FLS was also cloned using cDNA of longan leaves. Results ofbioinformatical analysis showed that the three sequences were highly homologouswith PAL, F3H and FLS respectively. The prediction results of Dlpal and Dlf3hcoding sequences showed that there were the conserved domains of PAL and F3H. Itwas inferred that Dlpal, Dlf3h and Dlfls were structural genes in longan leaves, andinvolved the processing of flavonoids biosynthsis.The molecular weight of Dlpal coded by Dlpal cDNA is306.4KD, the protein ishydrophilici and belonged to aromatic amino acid ammonia lyase containing PAL(PLN02457) and PAL-HAL conserved domain, and was similar with PAL and HAL.Transmembrane structure of Dlpal is outside-in. The Dlpal is synthesized onEndoplasmic reticulum and catalyzed phenylpropanoids. The molecular weight of Dlf3h coded by Dlf3h cDNA is45.3KD. The protein ishydrophilici and located on endoplasmic reticulum. Transmembrane structure ofDlf3h is outside-in and inside-out.3) The upstream sequences of CHI、CHS、PAL were cloned by TAIL-PCR kit,and the upstream sequences of F3H was cloned by I-PCR. The sequences CHI-P,CHS-P, PAL-P and F3H-P contained the promoter sequences of CHI, CHS, PAL andF3H each other, and were analyzed by PlantCare. There are light responsed elementsin all5sequences, such as SP1, Box4, L-box and so on. The results showed that thereare complex light responsed expression models in those genes involved in flavonoidssynthesis, and the expression of PAL, CHI, CHS and F3H are light dependent.Except for the light responsed elements, there are some element related to planthormone. The results showed that there are abscisic acid responsed element inpromoter sequences of F3H, CHS and CHI, according to that, it was concluded thatF3H, CHS and CHI are coordinated expression and regulated by endogenoushormones. Besides. In addition, CHI-P contains gibberellin responsed elementsTCCACCT-motif, CHS-P and PAL-P contain MeJA responsed elementsCGTCA-motif, CHS-P contained salicylic acid responsed elements TCA-element.4) There were different expression model of Dlpal、CHI、CHS、Dlf3h、Dlfls in4cultivar varieties. Considering the results in chapter2, it was concluded that thosegenes’transcript level were positively related to rutin and quercetin contents.Therefore, the genes Dlpal, CHI, CHS, Dlf3h and Dlfls played key roles in flavonoidssysthesis.更多还原