| Chinensis narcissus is originated in China, which is unique in Chinese flowers. Narcissus belongs to Monocots clade, Amaryllidaceae family, which forms bulb during summer dormancy, and flower bud differentiation is completed during this period. Therefore, the knowedge of dormancy mechanism of narcissus will make it possible to regulate the flowering time. Many reports focus on the study of the internal and external factors that influence the narcissus dormancy mechanism, but the study about the molecular mechanism is still rare. This study mainly focuses on the function of two genes NFT1 and NEIN3 which are related to dormancy of narcissus and other plant. The followings are the main results:(1) The the expression of NFT1 has been found to be relevant to flowering and dormancy of narcissus. The result of in situ hybridization assay showed that: Similar to other homologous genes of FT in different plants, NFTl was expressed in vascular bundles of mature leaves (phloem, xylem parenchyma), the cells of the upper and lower epidermis, the apical meristem organization and flower bud during the differentiation period, but during the period of dormancy (Apr.-Jul.), FT is hardly expressed.(2) The promoter of NFTl was cloned through hiTAIL-PCR. The sequences of NFTl promoter contain 800bp. Bioinformatics analysis shows that the promoter region possesses a series of environmental response element, such as a photo-responsive element, high-temperature and low-temperature responsive element, thermal-promote element and stress response element, which is corresponding to the result that promoting flowering and breaking dormancy are effected by temperature and light factors may be related to the expression of NFT1 which was founded during our previously study.(3) NFT1 plays a role in breaking plant’s seed dormancy. Seed germination rate of ft-3 mutant cultured in 1/2MS medium is obviously lower than the wild type. Meanwhile, seed germination rate of 35S::NFT1 in ft-3 background is obviously higher than ft-3, but is still no more the wild type. These results indicate that the FT gene mutations may inhibit seed germination, and overexpression NFT1 can break seed dormancy.(4) NFT1 may have the similar functions with ethylene. The expression of NFT1 and ethylene content are closely related in narcissus. During the physiological dormancy stage, the expression of NFT1 and ethylene content level are extremely low, but once physiological dormancy is broke, both the expression of NFT1 and ethylene content reach the highest level. In order to get to know the relationship between NFT1 and ethylene, FT mutants, ethylene mutants and NFT1 transformants of Arabidopsis thaliana were used as experimental materials to discover the ethylene response regulatory pathways. In the absence of ethylene in the medium, the length of hypocotyl and root with 35S::NFT1 in Col transformant is always shorter than that of the wild type obviously under dark conditions or exposure to the light. The plants transgenetic with 35S::NFT1 in Col have lower sensitivity to ethylene than the wild-type significantly also. Under dark conditions, Arabidopsis thaliana knocked out ft-3 mutant has much longer hypocotyl than the wild-type, but hypocotyl length of 35 S::NFT1 in Col in ft-3 mutant is significantly lower than the wild type and ft-3 mutant, and root of 35S::NFT1 in ft-3 mutant is shorter than ft-3 mutant which also has lower sensitivity to ethylene than ft-3 mutant similar to the wild type. These results show that overexpression NFT1 can lead to the ethylene reaction even without exogenous ethylene conditions.(5) NEIN3 in narcissus may have the similar functions with EIN3. In the ethylene response regulatory pathways of Arabidopsis thaliana, EIN3 gene is located downstream of this pathway and play as a positive regulator of ethylene response. Otherwise, CTR1 which is located downstream of the pathway and play as a negative regulator of ethylene response. We transformed the homologous gene of EIN3 which is named NEIN3 in narcissus to wild-type and mutant ein3-1 of Arabidopsis thaliana respectively for constructing 35S over-expression transformant. Physiological experiment showed that hypocotyl length of 35S::NEIN3 in Col was much shorter than the wild type in 1/2MS medium under darkness, and the sensitivity to ethylene was also significantly lower than the wild type. When exposure to the light, the hypocotyl length of 35S::NEIN3 in Col was similarly much shorter than the wild type in 1/2MS medium, and the sensitivity to ethylene was also significantly lower than wild-type (Further mutant and revertant study should be needed). Ethylene plays a role in breaking plant’s dormancy and promotes seed germination. The type of ctrl-1 and 35S::NEIN3 in Col have much higher seed germination rates than the wild-type at 1/2MS medium. All these results show that NEIN3 in narcissus has the similar function with EIN3, which is a positive regulator of ethylene response reaction.In summary, the expression of NFT1 is closely related to dormancy and growth of narcissus, the expression level of NFT1 are very low during the physiological dormancy stage. Overexpression NFT1 can revert the phenotype of FT mutant which has the low seed germination rate, and also can break seed dormancy; Overexpression NFT1 can lead to the ethylene reaction, especially in the light, the root length of Arabidopsis thaliana was suppressed; NEIN3 in narcissus has the similar function with EIN3, which is a positive regulator of ethylene response reaction. The phenotype of ethylene response reaction which germination rate is increased and root length is inhibited was showed when overexpression NEIN3 in Arabidopsis thaliana. Therefore, the results of this research can provide some references to further study the dormancy mechanism of Chinese narcissus and other plants. |
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