The Circadian Regulation Of LoPRI In Linalool Emission Of Lilium 'siberia'

As aperennial herb,Lilium 'siberia' plays an important role in cut flower markets in the world because of the various colors,big flower type and rich fragrance.Lilium 'Siberia' is one of commonest cultural Lilium Oriental hybrids,and can emit strong free aroma volatiles mainly including linalool.In plant cells,the glycosidase can control the transformation from the presence of glycosidically bound aroma precursors to free aroma volatiles.In this research,we use the Lilium ‘Siberia' cut flowers as material to clone LoPRI and many methods such as prokaryotic expression,activity analysis of recombinant protein in vitro,qRT-PCR,GC-MS and UPLC-Q-TOF-MS.We explore the light in the regulation of LoPRI and linalool emission of Lilium ‘Siberia'.These results will shed some light on the theoretical basis for molecular mechanisms on the formation and rhythmical fragrance emission in Lilium.The main results:1.The primeverosidase gene LoPRI was cloned by RT-PCR method.The full-length cDNA of LoPRI was 1482 bp encoding 493 amino acid residues with calculated molecular weight of 64.47 K Da,and pI was 5.28.LoPRI was a member of glycoside hydrolase family 1 which was consisted of two special structural domains: NEP region and ITENG region.2.The prokaryotic expression vector full-length of LoPRI was constructed.Obtained recombinant LoPRI produced in Escherichia colli almost specificly catalyzed the formation of abundant linalool,which was identified by SPEM and GC-MS,from glycosidically bound aroma precursors in Lilium flowers.It was only ?-primeveroside-linalool complex by UPLC-Q-TOF-MS analysis from linalool glycosidically bound aroma precursors of Lilium 'Siberia'.Thus LoPRI was surely primeverosidase in Lilium 'Siberia',which catalyzes the glucosidic bond from ?-primeveroside-linalool complex to free linalool.3.The expression levels of LoPRI in different tissues and during flower development were analyzed with qRT-PCR.The results revealed that LoPRI expression mainly concentrated on petal and sepal,following with root and leaf,and was detected at lower level in bulb.During flower development,the LoPRI expression level was different which was highest on blooming stage and lowest on fade stage.At 72 h normal condition(12 h light/12 h dark)and continuous light condition,the expression of PRI remained rhythmical,which was highest at daytime16 pm and lowest at night 24 pm to 4 am.But under continuous 72 h dark condition,the expression level reduced markedly,and the rhythm of LoPRI advanced.4.The emission level of free linalool and content of ?-primeveroside-linalool complex in different conditions were measured by GC-MS and recombinant LoPRI catalysis in vitro.The results revealed that?-primeveroside-linalool complex was lowest at daytime 16 pm and highest at night 4am,and the emission of free linalool was highest at daytime 16 pm and lowest at night 4am.Both maintain a dynamic balance and the rhythm just on the contrary.At 72 h continuous dark condition,the emission level of free linalool and content of ?-primeveroside-linalool complex both reduced markedly.These suggest that light plays an important role on the content of free and bound linalool,and LoPRI is the key gene in the circadian regulation of linalool emission.