Molecular cloning and functional characterization of the monoterpene synthase f-subfamily in Phalaenopsis bellina

Abstract

Plant secondary metabolites are the major nature products and terpenoid is the largest group among all Terpenoids play many roles in plants such as attracting pollinators and against herbivores or pathogenic bacteria Phalaenopsis bellina is a scented Phalaenopsis orchid with charming floral fragrance Previous studies have shown that the major floral compounds of P bellina are monoterpenes including geraniol linalool and their derivatives To clone the terpenoid synthase (TPS) from P bellina we used known TPS genes of other plants to search the genome database of P equestris Thirteen monoterpene synthase-like (MTPS-like) genes in P equestris were identified Among them four genes were less than 1000 bp and were excluded from the analysis Phylogenetic analysis of rest nine putative orchid TPS genes indicated that six genes were claded in angiosperm MTPS specific clade (TPS-b) and three were in the TPS-f subfamily The six TPS-b genes were annotated as either (?)-terpineol or (?)-pinene synthase and the three TPS-f genes were annotated as linalool synthase and designated PeMTPS6 PeMTPS7 and PeMTPS8 Gene expression analysis indicated that only PeMTPS7 and PeMTPS8 were expressed in the scented P bellina Rapid amplification of cDNA ends (RACE) was used to isolate the full length cDNA sequences of PeMTPS7 and PeMTPS8 in P bellina and named as PbMTPS7 and PbMTPS8 The temporal expression of PbMTPS7 indicated that it was expressed from five days before anthesis (D-5) to ten days post anthesis (D+10) with the highest expression level on three days post (D+3) and five days post (D+5) anthesis PbMTPS8 also showed high expression on D+3 and D+5 Spatially PbMTPS7 was not only expressed in the flower but also in the leaf and the expression level was up to a 4-fold higher level as compared to that in flower The spatial expression of PbMTPS8 indicated that expression level in vegetative organs was much lower than in flower Overall the expression level of PbMTPS8 was lower than PbMTPS7 In addition the expression patterns of PbMTPS7 and PbMTPS8 showed positive correlation with the volatile emission pattern of P bellina For enzyme functional assay both PbMTPS7 and PbMTPS8 were ectopically expressed in E coli were performed PbMTPS7 could use geranyl diphosphate (GDP) as a substrate for producing (β)-cis-ocimene and linalool while PbMTPS8 used GDP as a substrate for producing linalool Neither farnesyl diphosphate (FDP) nor geranylgeranyl diphosphate (GGDP) could be catalyzed by the two enzymes as substrates Moreover according to biosynthesis pathway of monoterpene in plants both PbMTPS7 and PbMTPS8 were expected to be localized in the chloroplast but they lack of typical transit peptide Thus subcellular localization prediction softwares could not precisely predict their subcellular localization For this green fluorescent protein was fused at either N- or C- terminus of both PbMTPS7 and PbMTPS8 and indicated that both PbMTPS7 and PbMTPS8 were localized in the chloroplast Taken together the knowledge gained from this study may lead to a better understanding of the monoterpene biosynthesis in P bellina

Date of Award	2014 Aug 27
Original language	English
Supervisor	Hong-Hwa Chen (Supervisor)