Functional Analysis Of Flower-specific Chimeric Promoters And Genetic Transformation Of Lilium ACO RNAi

Lily （Lilium spp.）, is one of the five most famous cutting flowers in the world. It is also anethylene-sensitive plant species. Senescence of flowers is stimulated by ethylene in manyornamental plants. Down-regulation or knock-out of the key genes related to ethylenebiosynthesis （ACO gene） by RNA interference （RNAi） technology would delay the flowersenescence and extend the vase life of cut flower. ACO genes are regulated in a tissue-specificmanner during flower senescence. In the previous work, we isolated an enhancedflower-specific promoter to replace of35S promoter to drive ACO RNAi vector with aim tominimize negative effects, we therefore constructed four chimeric promoters （p35S-PCHS-Ω,p35S-LCHS-Ω, pOCS-PCHS-Ω and pOCS-LCHS-Ω） fusion vectors and transformed withTorenia fournieri. In the current study, we examined the levels and tissue specificity of thesefour chimeric promoter fusion vectors in transgenic Torenia fournieri. Due to the relativelylow frequencies in stable transformations and many chimeras occurred in the regeneratedplants, we established an efficient regeneration system via somatic embryogenesis in lily.Finally, we introduced ACO RNAi vector into lily by Agrobacterium-mediated transformationand microprojectile bombardment. The main results are described as follows.1. Based on the preliminary studies, we further analysis the roles of four chimericpromoters （p35S-PCHS-Ω, p35S-LCHS-Ω, pOCS-PCHS-Ω and pOCS-LCHS-Ω）. Weexamined the levels and tissue specificity of GUS expression in transgenic Torenia fournieriusing histochemical staining, fluorometric assay and qRT-PCR. The results showed that:p35S-PCHS-Ω and p35S-LCHS-Ω displayed strongly constitutive GUS expression in alltissues, especially in colored corollas （p35S-PCHS-Ω） or in colored corollas and roots（p35S-LCHS-Ω）. pOCS-PCHS-Ω showed stronger GUS expression in colored corollas than inother tissues but expression was weaker than that of p35S-PCHS-Ω. pOCS-LCHS-Ω droveGUS in colored corollas but also in roots. Among the four chimeric promoters,pOCS-PCHS-Ω exhibited enhanced flower-specific expression and can replace35S promoterto drive ACO RNAi vector with minimal negative effects for transgenic enhancement of thevase life of lily.2. We constructed two ACO RNAi vector under the control of35S and the enhanced flower-specific promoter pOCS-PCHS-Ω, respectively. We cloned319bp ACO fragmentisolated from Orential hybrid lily ‘Sorbonne’ into specific sites （Xho I/EcoR I and Xha I/Cla I） of the interference vector pKANNIBAL, to generate pK-ACO-S-A RNAi vector, whichcontained a hairpin structure droved by35S promoter. Subsequently, pK-ACO-S-A subcoledinto the intermediate vector pAR-CAM. Eventually, the binary plant expression vectorpCAMBIA1301-35S-RNAi-ACO was constructed and introduced into Agrobacterium strainEHA105. To minimize potential adverse effects that can be present in transgenic plantstransformed with the35S promoter to control ACO RNAi vector expression, we used theenhanced flower-specific promoter pOCS-PCHS-Ω to replace35S promoter and constructedthe ACO RNAi plant expression vector pCAMBIA1301-OPCHS-RNAi-ACO.3. W established a regeneration system via somatic embryogenesis in lily. Filaments,pedicels, styles, ovaries and anthers of three cultivars: Orential hybrid lily ‘Sorbonne’,Oriental hybrid lily ‘Bernini’, and Oriental×Trumpet hybrid lily ‘Robina’ were used asexplants to identify and optimize the parameters influencing somatic embryogenesis. Theresults showed that the highest numbers of embryogenic cultures were induced from filaments（Sorbonne22.79%, Bernini16.51%, Robina17.77%）. The youngest developmental filaments（stagesⅠ） had the highest embryogenic response in each genotype, showing3–4-foldincreases compared with stageⅤ explants. Of the three regions of the filament used asexplants, only the basal ones produced embryogenic cultures. The highest embryogenic callusproliferation frequency was obtained using MS medium supplemented with2mg L^-1PIC and60.0g L^-1sucrose. For plant regeneration, these cultures produced the highest numbers ofplantlets when transferred to hormone-free MS medium with1g L^-1AC and30g L^-1sucrose.Histological and SEM observations of the various stages of somatic embryo developmentdemonstrated four typical stages: globular stages, heart-shaped stages, torpedo stages andcotyledonary stages.4. We introduced the ACO RNAi vector （pCAMBIA1301-35S-RNAi-ACO） into Orentialhybrid lily ‘Sorbonne’ genome by Agrobacterium-mediated transformation andmicroprojectile bombardment. When transformed, the critical concentration of hygromycinselection was25mg L^-1. Hygromycin-resistant lily plants were analyzed by PCR, Southernblot analysis and histochemical staining assay, which further proved that the ACO RNAivector （pCAMBIA1301-35S-RNAi-ACO） was introduced into Orential hybrid lily‘Sorbonne’ genome. We eventually obtained three transgenic lines of lily.