| 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. |
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