Functions Of Monoterpene Synthase In Lilium ‘Sorbonne’ And‘Red Life’ Based On The Differences Of Floral Fragrance

Lily is rich in floral fragrance.Most oriental lilies and their hybrids have strong fragrance,while Asiatic lilies and their hybrids have no fragrance or light fragrance.This extreme difference reduces the commercial value of lilies to a certain extent.Therefore,improving floral fragrance is an important goal of lily floral fragrance breeding.In this study,based on the differences of aroma components between flavor ’Sorbonne’and unscented ’Red Life’,we explored the functions of five TPS genes.The test results are as follows:The volatile compounds in ’Sorbonne’(38 kinds)were more than that in ’Red Life’(21 kinds).The total amount of volatile compounds released from ’Sorbonne’ were 18 times of that of ’Red Life’.The highest relative content of monoterpenoid is(E)-β-ocimene in ’Sorbonne’ and limonene in ’Red Life’.The biological information of five TPS proteins cloned from’ Sorbonne’ and ’Red Life’ was analyzed.It was found that the amino acid number of the five TPS proteins was between 548 and 590 and their molecular weight was about 65 k Da.They are both acidic proteins and hydrophilic proteins,and all of them are stable proteins except Lrl TPS-3.The five TPSs have no signal peptide but a chloroplast transit peptide.And the five TPSs have abundant α-helices and two structural domains.The important difference between them is mainly at the junction of the two structural domains.In the sequence,compared with Lso TPS-1 and Lrl TPS-1,Lso TPS-2inserted four amino acids,and another four amino acids were mutated;Lso TPS-3 and Lrl TPS-3 deleted 38 amino acids,another 22 amino acid sites were mutated in Lso TPS-3,and another 25 amino acid positions were mutated in Lrl TPS-3.The prokaryotic expression vector p ET32a-LTPS with His-tag was constructed.After inducing expression,it was found that the protein expressed by p ET32a-LTPS containing transit peptide was easy to form inclusion bodies and existed in the precipitate.Through optimization of expression conditions,soluble recombination protein could not be obtained.After that,the transit peptide was shortened to construct recombinant proteins p ET32a-(-)LTPS and p ET28a-(-)LTPS,which were expressed in prokaryotic expression system and purified to obtain soluble target protein.The enzyme activity of the purified LTPS proteins was detected by GC-MS.It was found that both Lso TPS-1 and Lrl TPS-1 could catalyze the formation of the main product ocimene with GPP,while Lso TPS-2,Lso TPS-3 and Lrl TPS-3 product α-pinene and limonene are produced with GPP.