Expression Vector Construction And Transformation Of Flowering And Flower Color Genes Of Zhangzhou Narcissus(Narcissus Tazetta Var.Chinensis)

Chinese narcissus (Narcissus tazetta var.Chinensis), a variety of French narcissus, is subordinate to Amaryllidaceae and mainly produced in Shanghai, Zhejiang and Fujian, Fujian is the best known with its highly ornamental and economic value. Chinese narcissus is triploid and difficult to breed new varieties through a natural or artificial hybridization. Limited species affect the product of Chinese narcissus. Currently, single species, low propagation speed and virus accumulation of narcissus have led to a decline in quality and cultivar degeneration, hindering the development of Chinese narcissus production. So, it is important to develop some new chinese narcissus species. Molecular biology and plant genetic engineering offer a new effective way to cultivar breeding for Chinese narcissus. In this study, Zhangzhou narcissus was used as the material. The flowering time genes API and FT, the key genes FLS and ANS controling flower colour in flavonoid biosynthesis pathway were cloned. Plant expression vectors pCAMBIA1301-NtFT, pCAMBIA1301-NtAP1,pC2300-35S-NtFT-OCS,pC2300-35S-NtAP1-OCS,pC2300-35S-NtANS-OCS were constructed. pBI121-FLSRNAi, pC2300-35S-NtFT-OCS and pC2300-35S-NtAP1-OCS were transferred into tobacco.Vectors pCAMBIA1301-NtFT and pCAMBIA1301-NtAP1 were transferred into Zhangzhou narcissus by Agrobacterium-mediated method. Plant expression vectors: pBI121-FLSRNAi and pC2300-35S-NtANS-OCS were also constructed and injected into narcissus flower petals and corona with Agrobacterium-mediated transient expression method. The results will provide technical basis and theoretical reference for the breed improvement of Chinese narcissus. Main results are as follows:1. The total RNA from zhangzhou daffodils flower bud and leaf was extracted and reverse transcribed into cDNA. A pair of specific primers were designed and synthesized according to the reported cDNA sequence of the FT,AP1,FLS,ANS genes. Cloned the key genes API, FT in Control flowering pathway and the key genes FLS, ANS in flavonoid pathway by PCR, Constructed plant expression Vectors pCAMBIA1301-NtFT,pCAMBIA1301-NtAP1,pC2300-35S-NtFT-OCS,pC2300-35S-NtAP1-OCS, pC2300-35S-NtANS-OCS, pBI121-FLS RNAi.Plant expression vectors pC2300-35S-NtFT-OCS and pC2300-35S-NtAPl-OCS were Transferred into tobacco by Agrobacterium mediated. Vectors pCAMBIA1301-NtFT and pCAMBIA1301-NtAP1 were transferred into zhangzhou daffodils by Agrobacterium mediated.2. Plant expression vectors pC2300-35S-NtFT-OCS, pC2300-35S-NtAP1-OCS were transferred into tobacco through Agrobacterium mediated, acquiring 31 resistant plants. Then, resistant plants were verified by PCR and Real-time quantitative PCR, Which Showed that the resistant plants were transgenic plants, They flowered earlier 10 days than non transgenic tobacco plants. Transgenic tobacco plants growed weaker than non transgenic tobacco plants.3. Plant expression vectors pCAMBIA1301-NtFT and pCAMBIA1301-NtAP1 were transferred to zhangzhou daffodils by Agrobacterium-mediated. Getting 3 resistant zhangzhou daffodils plants preliminarily.4. The GUS gene was injected into the narcissus flower petals and deputy crown by Agrobacterium mediated transient expression method, To explore whether this method was feasible. The results showed that the method was feasible. Thus Plant expression vectors pBI121-FLS RNAi, pC2300-35S-NtANS-OCS were injected into the narcissus flower petals and deputy crown by Agrobacterium mediated transient expression method. We observed that narcissus flower petals and deputy crown color have changed.