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Flower development of Lilium longiflorum - The Lilium information ...

Flower development of Lilium longiflorum - The Lilium information ...

Chapter 3 A B Figure 4.

Chapter 3 A B Figure 4. Phenotype of a T1 Arabidopsis overexpressing LLSEP3. A. Plants were very weak, showing very early flowering and curled leaves. B. Close-up of plant shown in picture A. Among 25 transgenic plants analysed, 15 plants showed this phenotype and were followed up to the next generation, which presented this phenotype in most of the plants. 1 2 3 4 5 6 LLSEP3 NPTII Figure 5. Northern blot analysis of representative early flowering T1 Arabidopsis plants overexpressing LLSEP3. Approximately 10 µg of total RNA were loaded in each lane. Upper panel shows hybridisation with the LLSEP3 probe. The blot was stripped and re-hybridised with the NPTII probe, shown in the lower panel. Lane 1 was loaded with RNA extracted from a non-transgenic plant, lanes 2 to 5 are from plants showing phenotypic alterations whereas lane 6 is derived from an Arabidopsis resembling its wild type. Altogether, the sequence similarity between LLSEP3 and E functional genes, the active expression through floral development and its persistence in mature floral organs, and the capability of promoting early flowering when overexpressed in the heterologous species Arabidopsis indicated that LLSEP3 can potentially be the functional orthologue of SEP3 in Lilium longiflorum. 42

Material and Methods Plant material Isolation and Characterisation of LLSEP3 Mature floral organs, developing buds and leaves were collected from lily (Lilium longiflorum Thunb.) plants growing under greenhouse conditions and immersed immediately in liquid nitrogen. Tissues were kept at -80 o C until use. After seed vernalization for 3 days at 4 o C, wild-type Arabidopsis thaliana plants, ecotype Columbia (Col), were kept in a growth chamber under long day conditions at 23 o C until flowering. The first inflorescence stems were pruned to induce formation of lateral inflorescences for transformation. LLSEP3 probe synthesis, cDNA library construction and screening A homologous SEP3 probe from lily for screening a cDNA library was produced by RT-PCR using total RNA derived from 1-cm floral buds and degenerated oligonucleotides designed from conserved regions between the end of the MADS-box and the carboxy-termini of SEP3 homologous sequences available in the GenBank database. The first strand synthesis reaction was primed with an oligo-d(T)12-18 supplied with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) and followed by a gene-specific amplification with the oligonucleotides 5’-CTC TAC GAG TTC TGC AGC-3’ and 5’-GMT CCC ACA CYT GCT GC-3’, as forward and reverse primers using an annealing temperature of 45 o C in the PCR. A 391-bp fragment was amplified, cloned into pGEM-T-easy vector (Promega, Madison, WI) and sequenced. High sequence homology could be observed between this clone and other putative SEP3 homologues and therefore it was used as a probe for cDNA library screening. A developing floral bud (1 to 3.5 cm) cDNA library from Lilium longiflorum was synthesised using the ZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene, La Jolla, CA) with 5 µg of pooled poly(A) + RNA. Fragments ranging from 1 to 1.5 kb were selected for unidirectional insertion in Uni-ZAP XR phage vector. The cDNA clones were rescued into pBluescript SK + by in vivo excision using the ExAssist helper phage. About 50,000 pfu were screened on nylon membranes at 60 o C with the 391-bp homologous probe. 43

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