大葉蝴蝶蘭(Phalaenopsis bellina)香味生合成及其相關基因之研究

Floral scent is a key modulating factor in plant-insect interactions and thus plays a central role in successful pollination. Orchidaceae, containing more than 25,000 species, is one of the largest monocotyledon families. Pollinators have played an important role in driving floral diversification in orchids, which has obviously been advantageous for the evolution of this highly successful family. However, the biochemistry of fragrance production and the mechanisms that regulate scent emission have not been well studied. We investigate how flower fragrance is generated in certain Phalaenopsis orchids by identifying floral expressed-sequence-tags (ESTs) and deducing the floral scent biosynthesis pathway of Phalaneopsis bellina with bioinformatics analysis. The major chemical components in the P. bellina flower are found to be monoterpenoid, benzenoid, and phenylpropanoid as revealed by gas chromatography-mass spectrometry. The major volatiles were found to be monoterpenes of geraniol, linalool, and their derivatives, and maximally emitted from day 5 post-anthesis to day 7 post-anthesis. The encoded enzymes collection in the biosynthetic pathway from glycerolaldehyde-3-phosphate (G3P) to geraniol and linalool are recognized through data mining of the P. bellina floral EST database (dbEST). Transcripts preferentially expressed in P. bellina include those encoding lipoxygenase, epimerase, diacylglycerol kinase and geranyl diphosphate synthase (GDPS). EST filtering results showed that transcripts encoding signal transduction and Myb transcription factors and methyltransferase, in addition to those for scent biosynthesis, are detected by in silico hybridization of the P. bellina unigene database against those of the scentless species, rice and Arabidopsis. Altogether, we pinpoint 66% of the biosynthetic steps from G3P to geraniol, linalool and their derivatives. We then focused on the key enzymes in this pathway, the geranyl diphosphate synthase (GDPS) which catalyzes the formation of GDP for further synthesis of geraniol, linalool and their derivatives. The novel PbGDPS_SSU (GDPS small subunit) enzyme lack the conserved aspartate-rich motif DD(X)2-4D, possesses a dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP) and functional as a homodimer. Homology modelling and site-direct mutagenesis of PbGDPS_SSU indicate that the Glu-rich motif may provide a binding site for Mg2+ and catalyze the formation of prenyl products. Expression pattern result show the PbGDPS_SSU was flower specific and maximal PbGDPS expression is concomitant with maximal emission of monoterpenes on day 5 post-anthesis, thereby, we suggests that PbGDPS_SSU plays a crucial role in scent production/emission in orchid flowers. Furthermore, we also isolated the PbGDPS_LSU (GDPS_large subunit) whose sequence was similar to GGDPS and its function of scent precursor production was analyzed by using yeast two-hybrid analysis. The coexpressed proteins of both PbGDPS_SSU and PbGDPS_LSU showed the most prominent of prenyl transferase activities as compared to each subunit protein expressed alone. These results suggest that the heterodimeric PbGDPS_LSU/SSU may play a crucial role in monoterpene biosynthesis for P. bellina flower. In addition, several R2R3-type MYB transcription factors were isolated from the floral dbEST of P. bellina. These included PbMYB1 and PbMYB2 with an open reading frame of 202 a.a. and 141 a.a., respectively. Transcriptional levels of PbMYB1 and PbMYB2 reached the maximum on D+5 (day 5 post-anthesis), consistent with the fact of both maximal emission of monoterpenes as well as maximal expression of PbGDPS_SSU on D+5. It is possible that both PbMYB1 and PbMYB2 may be well correlated to the regulation of monoterpenes production in P. bellina flowers. In conclusion, these approaches demonstrate the opportunity to study floral scent in the non-model plants such as orchids, and moreover to accelerate molecular breeding in orchid industry.