【作者】 陈段芬；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 生态学， 2008， 博士

【摘要】 中国水仙(Narcissus tazetta var.chinensis)是世界著名的球根花卉,也是我国传统名花中出口创汇的重要种类。由于中国水仙是同源三倍体,利用传统的杂交和实生选种方法产生新品种存在极大困难,加之野生资源高度匮乏,致使中国水仙品种单一成为我国水仙产业发展的瓶颈问题。克隆与中国水仙花型和花色发育相关的功能基因,建立优化的中国水仙遗传转化体系,不仅可以在一定程度上揭示中国水仙花型和花色的变异机理,还可为利用植物基因工程改良中国水仙的观赏性状提供理论依据和技术支持。本研究对中国水仙的两个MADS-box基因(NTMADS1和NtMADS3)、两个花色发育基因(NTPDS1和NTZDS1)和遗传转化体系进行了研究,主要结论如下:1、利用RT-PCR技术,从中国水仙‘玉玲珑’幼嫩花序中克隆到NTMADS1基因,序列分析表明,NTMADS1 cDNA片段总长为879 bp,含有完整的开放阅读框架,编码230个氨基酸。与拟南芥MADS-box基因家族的系统进化树分析表明,该基因属于AG亚族的C类功能基因。利用RT-PCR方法,研究了NTMADS1基因在开花期的组织特异表达模式,发现NTMADS1基因只在中国水仙花中表达,且丰度极高;在花的雄蕊中表达量最高,其次为副冠、雌蕊,在花瓣中几乎未检测到该基因表达。以pBI121为载体,构建了该基因的正义表达载体,通过农杆菌介导的叶盘法和蘸花法,将构建好的基因分别在烟草和拟南芥中异位表达。通过对转基因植株的表型观察发现,转NTMADS1基因的拟南芥植株开花期明显早于野生型,且花型出现显著变化,如雄蕊心皮状、花瓣全部或部分退化、顶部茎生叶出现花状结构等。转基因烟草则出现花冠裂片变短且边缘不规则、花筒开裂、花药退化等现象。初步表明NTMADS1参与了花型和花期的调控。2、利用RT-PCR和RACE技术,从中国水仙‘金盏银台’幼嫩花序中克隆到NtMADS3基因,序列分析表明, NtMADS3 cDNA总长为980 bp,含有一个726 bp的完整ORF,编码241个氨基酸。与拟南芥MADS-box基因家族的系统进化树分析表明,该基因与拟南芥(A. thaliana)的AGL6亲缘关系最近,属于E类功能基因。利用RT-PCR方法分析了该基因在开花期的组织特异表达模式,发现NtMADS3基因在中国水仙开花期的花、叶片、鳞片和根系中以及花的各个部位均有表达。将构建在pBI121载体上的NtMADS3基因在烟草和拟南芥中异位表达,通过对转基因植株表型观察发现,转NtMADS3基因拟南芥植株开花期明显早于野生型,但花型未出现显著变化;部分转基因植株出现植株矮小和莲座叶卷曲现象,但绝大部分表现正常。转基因烟草则出现花筒开裂、雄蕊退化或瓣化等现象。初步表明NtMADS3基因参与了花期和花型的调控。3、从中国水仙‘金盏银台’幼龄花序中克隆到中国水仙八氢番茄红素脱氢酶(NTPDS1)基因和ζ-胡萝卜素脱氢酶(NTZDS1)基因。序列分析表明,NTPDS1 cDNA片段总长为1 719 bp,含有完整的开放阅读框架,可编码含570个氨基酸;NTZDS1基因总长为1 899 bp,ORF可编码574个氨基酸。两个基因均在中国水仙的花中大量表达,且在花瓣和副冠中的表达量高于雄蕊和雌蕊;叶片和根中的表达量次之;在鳞片中表达量极少或检测不到表达。构建了两个基因的pBI121和pCAMBIA1300植物表达载体,并通过农杆菌将上述基因转入烟草和拟南芥中,得到了卡那抗性的再生植株。4、以中国水仙不同部位外植体进行了不定芽直接分化和愈伤组织诱导分化试验,结果表明,双鳞片外植体在MS0+6-BA 10.0 mg/L+NAA 1.0 mg/L+蔗糖40 g/L+琼脂7.5 g/L培养基上可以大量地直接再生,再生芽数量平均可达13~16个/cm双鳞片,比传统的鳞茎盘之间分化提高30~50%;鳞片、叶片、花葶及花部不同组织在适宜的配方中均可不同程度地诱导出愈伤组织,但以带花药的花丝诱导出的愈伤组织最好。筛选抗生素和抑菌抗生素浓度试验表明,花丝产生的愈伤组织的选择压为潮霉素15~20 mg/L;羧苄青霉素的抑菌浓度为250~300 mg/L。更多还原

【Abstract】 Narcissus tazetta var.chinensis is a world-renowned bulb flower and plays an important role in Chinese flower trade. As a homologous triploid plant, it is very difficult to create new varieties by traditional hybridization and seedling breeding methods. In addition, the wild Narcissus resources are extremely deficient in China, so varieties deficiency has become the bottleneck in Chinese Narcissus industry. Cloning genes related to flower colour and flower organ identity from N. tazetta var.chinensis and establishing optimized genetic transformation system can not only explain the variation mechanism of N. tazetta var.chinensis flower colour and flower organ identity to a certain extent, but also offer theoretic and technical support to improve or create the ornamental characters through plant genetic engineering.Two MADS-box genes(NTMADS1 and NtMADS3), two colour development related genes(NTPDS1 and NTZDS1) and genetic transformation system of N. tazetta var.chinensis were studied in this paper, the major findings are described as followings:1. A gene named as NTMADS1 was isolated from the flower alabastrums of N. tazetta var.chinensis by RT-PCR method. The cDNA is 879 bp in length with an open reading frame which is capable of encoding 230 amino acid. Compared with A. thaliana MADS-box genes family, phylogenetic tree analysis indicated that NTMADS1 belongs to AG subfamily of C class genes.The tissue-specific expression pattern was studied by RT-PCR method. The results revealed that the transcript of NTMADS1 was not detectable in vegetative tissues, but only in flowers. Within open flower organs, transcript levels were much higher in stamen and corona than in pistil and barely detectable in petals.To explore the role of NTMADS1 in flower organ identity, the expression vector of pBI121-NTMADS1 with sense orientation was constructed. Transgenic A. thaliana and N. tabacum with pBI121-CaMV35S-sense NTMADS1 were generated via Agrobacterium tumefaciens-mediated flower-dipping and leaf disk transformation method. Ectopic expression of NTMADS1 in transgenic A. thaliana resulted early flowering, curly leaves, carpel-like stamen, obsolete or absent petals and flower-like stem leaf. Similarly, N. tabacum over-expressed NTMADS1 appeared shorted, dehiscent and deformed tubular corolla and absent or petal-like stamen. All of this indicated that NTMADS1 plays an essential role in the regulation and control of flower organ identity and flowering time.2. By means of RT-PCR and RACE method, a new gene named as NtMADS3 was isolated from the flower buds of N. tazetta var.chinensis. The sequence analysis showed that this cDNA is 980 bp in length with a 726 bp ORF encoding 241 amino acid. Phylogenetic tree analysis indicated that NtMADS3 is most similar to AGL6 of A. thaliana and was classified into E class function genes. The results of tissue-specific expression pattern revealed that the transcript of NtMADS3 was detectable not only in any whorl of flower but also in vegetative organs.pBI121-CaMV35S-sense NtMADS3 was transferred into A. thaliana and N. tabacum via Agrobacterium tumefaciens-mediated method. Ectopic expression of NtMADS3 in transgenic A. thaliana caused early flowering and curly leaves in several transgenic plants, but many transgenic plants had no change in phenotypes. Whereas, N. tabacum over-expressed NtMADS3 appeared shorted, dehiscent and deformed tubular corolla and absent or petal-like stamen, which is similar to the phenotypes of transgenic N. tabacum with NTMADS1gene. Above phenomenons indicated preliminarily that NtMADS3 perhanps involved the regulation of flowering time and flower organ identity.3. Two dehydrogenase genes, named as NTPDS1 and NTZDS1, were obtained from the alabastrums of N. tazetta var.chinensis by RT-PCR method. Sequence analysis showed that NTPDS1 is 1 719 bp in length with an ORF encoding 570 amino acid, and NTZDS1 is 1 899 bp in length with an ORF encoding 574 amino acid. Tissue-specific expression model revealed that the expression level of NTPDS1 and NTZDS1 was extremely high in flowers, especially in petals and corona. The transcript was detectable in leaves and roots as well, but barely exist in scale leaves. The plant expression vectors were constructed and two genes were transferred into A. thaliana and N. tabacum, antikanamycin plants were obtained.4. The trials of adventive bud regeneration directly and callus induction was conducted to select out the optimal explants and formula. The results revealed that double scale can produce 13~16 adventive buds per centimeter in MS medium added 10.0 mg/L 6-BA, 1.0 mg/ L NAA, 40 g/L sugar and 7.5 g/L agar. Scale, leaves, scape and different tissue of flower can be induced to produce callus in appropriate medium, but the filament with anther was the optimized explant. The experiment of antibiotic showed the optimized hygromycin concentration was 15~20 mg/L and the recommendated carbenicillin was 250~300 mg/L for callus from filament with anther.更多还原