Cloning And Characterization Of An LiBSMT Gene Involved In Methyl Benzoate Production In Lilium

Floral scent is one of the most important characteristics of ornamental plants, and is a research focus in recent years. The main constituents of floral scent are terpenoids, benzenoids/phenylpropanoids and fatty acid derivatives. Methyl benzoate is the main substance of benzenoids and important component in Lilinm, which was synthesized by benzenoid carboxyl methyltransferases. This class of enzymes and genes has been well studied in model plants, but has not been reported in Lilium. In order to clarify the emission and regulation mechanism of main volatile methyl benzoate, Lilium’Yelloween’ of OT hybrid cultivar was chosen as experimental material, and the space-time emission of methyl benzoate was analyzed by static headspace adsorption and gas chromatography-mass spectrometry (GC/MS) coupled to automatic thermal desorption (ATD). Using homologous cloning method and RACE, we obtained the final step synthesis gene of methyl benzoate. The expression patterns and function of the protein encoded by the gene were then analyzed. The main results are as follows:1. Release of methyl benzoate performed certain space-time patterns:petals are major resource; emission of methyl benzoate increased and then decreased during flowers development; its emission is obviously different during a day, and emitted higher during 3 PM to 9 PM, while emitted lower in other time.2. We obtained a benzoic acid/salicylic acid carboxyl methyltransferase gene, which was the final step in the metabolic pathway of methyl benzoate, named LiBSMT, and GenBank accession number was KJ755672. The gene has an open reading frame of 1083 bp and encodes a protein of 360 amino acids with a calculated molecular mass of 41.05 kDa. Sequence alignment of LiBSMT revealed 40-50% similarity with other known benzenoid carboxyl methyltransferases in other plant species, and phylogenetic analyses showed homology to BSMT of Oryza sativa.3. Quantitative real-time PCR results showed that LiBSMT gene expression was consistent with the emission of methyl benzoate, which also performed certain space-time patterns. LiBSMT was preferentially expressed in petals; during the lifespan of Lilium ’Yelloween’, the expression of LiBSMT in petals was developmental regulated; circadian rhythm analysis indicated that LiBSMT gene expression level increased in the afternoon; LiBSMT expressed relatively high in Lilium sulphureum, Lilium regale, Lilium’Yelloween’ and Lilium’Conca d’Or’, while little or no transcript level could be detected in Lilium ’Siberia’, Lilium tsingtauense, Lilium’Sorbonne’, Lilium Tresor’and Lilium’Ceb Dazzle’. It can be indicated that LiBSMT gene may be involved in the regulation of the release of methyl benzoate.4. Heterologous expression of LiBSMT gene in Escherichia coli yielded an enzyme responsible for formation of methyl benzoate. Using prokaryotic expression technology, we constructed a recombinant expression vector and expressed a protein of about 40 kDa in Escherichia coli, which was identical to the predicted molecular mass. The growth medium of E. coli cultures was supplied with substrates and then extracted with hexane. After analyzing the extracts with GC/MS, there were methyl benzoate and methyl salicylate generated, and the content of methyl benzoate was higher.This experiment comprehensively studied the emission and regulation mechanism of methyl benzoate in lily, and provided information regarding floral scent research and scent-related breeding in Lilium.