| Summary: | 博士 === 國立中興大學 === 農業生物科技學研究所 === 90 === Abstract
Although lilies are among the most important plants in flower markets around the world, relatively little research has been conducted on lily flower development. In this research, four MADS box genes and one protein kinase gene were cloned and characterized from lily (Lilium longiflorum).
LMADS1, with sequence homology to the AP3 family of genes, contains complete consensus sequence of the paleoAP3 motif found in the AP3 family of genes from the low eudicot, magnolid dicot and monocot species. Ectopic expression of LMADS1 cDNA truncated with the MADS box domain in Arabidopsis generated the ap3-like dominant negative mutation in which the petals were converted into sepal-like structures and the stamens were converted into carpel-like structures. Different from other B functional genes, LMADS1 is able to form homodimers through yeast two-hybrid analysis. These results indicated that LMADS1 represents an ancestral form of the B function gene, which retains the ability to form homodimers in regulating petal and stamen development in lily (Charper 2).
Based on amino acid sequence, three motifs in C-terminal region of LMADS1 may involve in the formation of homodimers for LMADS1. The ability for LMADS1 to form homodimers was decreased once C-terminal paleoAP3 motif was deleted. When three motifs in C-terminal region were deleted, the ability for LMADS1 to form homodimers was completely abolished. Furthermore, Arabidopsis AP3 was able to form homodimers once C-terminal region was replaced by that of LMADS1. This result indicated that three motifs in C-terminal region of LMADS1 were responsible for the formation of homodimers in ancestral form of the B function gene (Charper 3).
Transgenic Arabidopsis ectopically expressed two lily MADS box genes LMADS2 or LMADS3 showed similar novel phenotypes by flowering extremely early and losing inflorescence indeterminacy. The expression of flowering time genes CO, LD, FT and AP1 was significantly up-regulated in these transgenic plants. Ectopically expressed these two genes rescued late-flowering phenotype for co, gi but not for ft and fwa. In vitro binding assay indicated that LMADS2 and LMADS3 proteins bound efficiently to CArG box region identified in the regulatory region of CO and FT. These data provided first evidence to support that early-flowering phenotype generated by ectopic expression of MADS box genes is due to the positive interaction between MADS box and flowering time genes in transgenic Arabidopsis (Charper 4).
Transgenic Arabidopsis plants ectopically expressed lily p70 ribosomal S6 kinases homologue LS6K1 significantly reduced the length of petals and stamens by inhibiting cell expansion. The expression of NAP, a downstream gene for AP3 and PI, was significantly upregulated by ectopic expression of LS6K1. Furthermore, oligopyrimidine tract sequences were identified in 5’ untranslated region of AP3, PI and SUP cDNA. This indicated that they were potentially encoded 5’TOP mRNAs and were translationally controlled by phosphoryated 40S ribosomal protein S6. This assumption was supported by the fact that GFP proceeded by these oligopyrimidine tract sequences was translationally regulated in human 293 cells in response to mitogen stimulation. These results revealed a novel role for p70 ribosomal S6 kinases in regulating petal and stamen growth and development by translational up-regulation of AP3, PI, and SUP (Charper 5).
Finally, LMADS4 showing high homology to the AGL2 subgroup of MADS box genes was isolated and characterized in lily. LMADS4 encodes a 246 amino acid protein that showed 61% identity and 73% similarity to cucumber AGL2 (CAGL2). The amount of LMADS4 mRNA detected in floral buds of different developmental stages, was higher that of in vegetative leaves, inflorescence and floral meristems , but was absent in stems. In flowers, similar to AGL2 and AGL4, LMADS4 was expressed in all four flower organs, sepal, petal, stamen and carpel. These results indicate that LMADS4 is a putative AGL2 homologue of lily and functions in regulating the flower formation as well as the floral initiation (Charper 6).
The characterization of genes in this study indicated that the MADS box genes also play important role in the flower development in monocots. These results should be very useful in understanding the mechanism controlling floral transition and floral formation among higher plant species.
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