【作者】 盛艳乐；

【导师】 陶俊； 周春华；

【作者基本信息】 扬州大学 ， 果树学， 2010， 硕士

【摘要】 三萜类化合物广泛存在于多种植物中,种类繁多,其中最常见的是五环三萜中的齐墩果烷型和乌苏烷型,其代表化合物分别是齐墩果酸(OA)和熊果酸(UA)及其衍生物。目前在柿(Diospyros kaki L.)中关于OA和UA的研究主要集中在叶片和柿蒂中,而柿果皮和果肉中尚未有研究。本论文以柿果实为试材,对32个品种(23个涩柿和9个甜柿)柿果肉OA和UA含量进行分析;选取代表品种(牛心柿和西村早生)测定柿果实不同发育阶段OA和UA的含量;并克隆柿子OA和UA等五环三萜合成相关基因(SQS、OSC)的全长或片段,研究柿果实不同发育阶段三萜合成相关基因的表达状况。主要研究结果如下:1.对32个品种柿果肉OA和UA含量进行分析。结果表明,不同品种之间,其果肉OA和UA含量差异很大。涩柿品种OA和UA的含量分别为0～88.57μg.g-1 FW和0～27.64μg.g-1 FW,甜柿品种OA和UA的含量分别为1.24～26.43μg.g-1 FW和0～12.23μg.g-1 FW。2.对代表品种(牛心柿和西村早生)柿果实不同部位、不同发育时期OA和UA含量进行分析。结果表明,柿果实不同部位中OA和UA的含量一般以果蒂最高,果皮次之,果肉最低。且OA和UA的含量随生长期不同而发生显著变化。一般果肉和果皮在果实发育初期,OA和UA的含量相对较高,而果蒂在果实成熟期OA和UA相对较高。而将涩柿和甜柿作比较,三个部位多数阶段OA和UA的含量都是甜柿高于涩柿。3.根据植物鲨烯合成酶(SQS)基因的保守区序列,设计1对PCR引物,采用RT-PCR技术,以柿果肉RNA为模板克隆出鲨烯合成酶基因的片段,在此基础上设计引物进行3’和5’-RACE,最后拼接出cDNA全长1467 bp,经过序列分析,其编码415个氨基酸。在GenBank中进行同源性检索的结果表明,该序列编码的氨基酸与其他植物鲨烯合成酶编码的氨基酸同源性较高,注册号为FJ687954。4.根据GenBank中其它植物2,3-氧化鲨烯环化酶(OSC)基因序列设计引物,以柿果肉RNA为模板模板,采用3’-RACE方法进行3’端片段的扩增得到cDNA 3’端部分序列。该cDNA3’端部分长522bp,包含一个298 bp的开放阅读框(ORF),3’端有224 bp的非翻译区(UTR)并具有完整的PolyA尾巴,编码98个氨基酸。5.对柿果肉不同发育阶段SQS基因和OSC基因相对表达水平研究表明,SQS和OSC基因的表达水平与柿果肉中OA和UA的合成相关性不明显。更多还原

【Abstract】 Triterpenoids, which include many varities, are widespread in plants. The most common compouds are oleanane and ursane-type pentacyclic triterpenoids. The representative compounds were oleanolic acid (OA) and ursolic acid (UA) and their derivatives. At present, the research of OA and UA in persimmon (Diospyros kaki L.) is mainly focused on leaves and pedicles, while OA and UA content in the peel and flesh of persimmon fruit has not been examined.In this study, persimmon fruits of 32 cultivars (23 astringent and 9 non-astringent) were used as materials, and OA and UA content in these fruits were determined. OA and UA content in typically selected cultivars‘Niuxinshi’and‘Nishimurawase’at different developmental stages were also analysised. The full-length or fragment of SQS and OSC genes were cloned, and invesgated the expression levels of these genes. Research results are found as follows:1. OA and UA contents in the flesh of 32 different cultivars were determined. Results showed that the contents of OA and UA in the fleshs of different persimmon cultivars were significantly different. The contents of OA and UA were 0~88.57μg.g-1 FW and 0 ~27.64μg.g-1 FW for astringent cultivars, respectively. While the contents of OA and UA were 1.24~26.43μg.g-1 FW and 0 ~12.23μg.g-1 FW for non-astringent cultivars, respectively.2. OA and UA contents in different parts of persimmon fruits and at different developmental stages of two representative cultivars‘Niuxinshi’and‘Nishimurawase’were analyzed. The data showed that the contents of OA and UA varied according to different parts of persimmon fruits measured, pedicle ranking first, next peel and then flesh. Moreover, the contents of OA and UA differed significantly with their growing periods. The contents of OA and UA in flesh and peel were comparatively high in the initial growing period, while that of pedicle in mature period were higher than other periods. If comparisons were made between astringent and non-astringent persimmons, the latter’s contents of OA and UA were higher than that of the former regardless of the three different parts and various growing periods.3. On the basis of conserved sequence of squalene synthase gene in other plants, a pair of PCR primers were designed to clone the fragment of squalene synthase gene in persimmon fruit with RNA as template by the support of RT-PCR techonology. Then, primers were designed for 3’ and 5’-RACE, and finally the 1467 bp long full-length cDNA was spliced. The gene encoded 415 amino acids by sequence analysis. The result of homology research in GenBank showed that amino acids encoded by this sequence and other squalene synthase gene -encoded ones were much homologous, and the registration number was FJ687954.4. On the basis of 2,3-oxidosqualene cyclase gene sequenc in other plants, primers were designed to amplied 3’ partial sequence of 2,3-oxidosqualene cyclases gene cDNA in persimmon fruit with RNA as template through 3’-RACE method. This fragment was 522bp long and encoded with 98 amino acids, with an ORF 298 bp long, UTR 224 bp long and a complete PolyA tail.5. The comparative expression researches of SQS and OSC genes in the flesh of persimmon fruit at different developmental stages indicated that SQS and OSC genes expressionswere not significantly correlated with the biosynthesis of OA and UA in persimmons flesh.更多还原