【作者】 江琳玉；

【导师】 潘东明；

【作者基本信息】 福建农林大学 ， 园林植物与观赏园艺， 2009， 硕士

【摘要】 中国水仙(Narcissus tazetta Lin.var.chinensis Roem)为石蒜科水仙属植物,多年生草本,是多花水仙的一个变种。福建漳州是中国水仙主要产地之一,漳州水仙于2008年获得国家地理标志驰名商标。目前,花色单一己成为制约中国水仙发展的主要障碍之一,因此,对中国水仙进行花色改良具有重大的意义。本研究以漳州水仙品种‘金盏银台’为材料,采用RT-PCR和RACE技术,克隆得出了中国水仙ZDS(ζ-胡萝卜素脱氢酶基因)cDNA全长序列和LYCE(番茄红素ε-环化酶基因)片段序列,并对ZDS进行原核表达研究,为下一步利用花色基因进行遗传转化奠定基础,以期为中国水仙花色遗传改良提供一条新途径。主要研究结果如下:1、提取中国水仙花瓣和副冠的总RNA,反转录为cDNA二链,并以此为模板克隆类胡萝卜素合成途径中的关键基因ZDS(登录号为:EU573238),ZDS全长2189 bp,其中包含69 bp5′非编码区,395 bp3′非编码区,1725 bp的编码区(编码574个氨基酸,分子量约为63.6 kDa)。通过BLAST工具对比发现,该ZDS氨基酸序列与黄水仙(CAA12062.1)、葡萄(CAO43021.1)和蜜柑(BAB68552.1)的ZDS基因cDNA的同源性都很高,分别为97%、85%和83%。2、将分离的中国水仙ZDS基因cDNA的编码区序列插入含有EcoRI和XhoI酶切位点的pET-28a原核表达载体上,并在大肠杆菌BL21细胞中进行原核表达,结果表明,该编码区片段在大肠杆菌中表达,分子量约为63.6 kDa的蛋白质,与理论值相符。3、采用RACE方法获得LYCE cDNA片段序列长1235 bp,编码371个氨基酸残基,其推导的氨基酸序列与胡萝卜(ABB52073.1)、柚子(AAX92679.1)、甜橙(AAS48096.1)和温州蜜桔(BAE93359.1)的氨基酸序列同源性分别为80%、76%、76%和75%。更多还原

【Abstract】 Narcissus tazetta L. var. chinensis Roem, a kind of perennial plant with beautiful color and sweet fragrance, belongs to the family Amaryllidaceae. Zhangzhou which is in FuJian province is the most important produce area of Chinese narcissus and the Zhangzhou narcissus have gotten a National Geographical Indication of well-known trademarks in 2008. But the simplex color have become a main obstacle for Chinese narcissus’s development. So it’s imperative to develop new varieties with modern breeding methods for the narcissus industry.In this research, we isolated the full length cDNA of Zeta-carotene desaturase gene (ZDS) and a partial sequence of Lycopene epsilon cyclase gene(LYCE) from Chinese narcissus petals by RT-PCR and RACE and tried to express ZDS in E.coli. This laid a foundation for its application on subsequent genetic transformation study, which aims to provide a new way to the genetic improvement of the color of Chinese narcissus.1、The RNA extracted from Narcissus flower petal was used for the isolation of ZDS by RT-PCR (GenBank accession number: EU573238). The results of analysis of the coding sequences of the genes demonstrated that the gene of ZDS is 2189bp long. It contains an ORF encoding 499 amino acid residues(Mw=63.6kDa), a 5’untranslated region of 69bp and a 3’untranslated region of 395bp. Compared with ZDSs of other plants, Chinese narcissus’s amino acid sequence had 97%, 85%, 83% identity with that of Narcissus pseudonarcissus, Vitis vinifera and Citrus unshiu.2、The ZDS gene cDNA was insert into the vevtor pET-28a degisted by EcoR I and Xoh I. The expression construct was transformed into BL21 and was indued. The result displayed that the ZDS was expressed in E.coli. And the SDS-PAGE experiments show that the actual value of ZDS molecular weight agrees well with the theoretical one.3、The partial sequence of LYCE isolated from Chinese narcissus by RACE is 1235bp long, encoding 371 amino acid residues. It’s amino acid sequence had 80%, 76%, 76%, 75% identity with that of Daucus carota, Citrus maxima, Citrus sinensis and Citrus unshiu.更多还原