The Roles Of MYC2 Transcription Factor In The Fragrance Biosynthesis Of Lilium 'Siberia'

Lilium ‘Siberia' is one of commonest cultural Lilium Oriental hybrids.As one of the world's most popular cut flowers,Lilium ‘Siberia' is famous for its intense fragrance,white petals and longevity.The role of Lo MYC2 transcription factor in the biosynthesis of floral volatiles and interaction with Lo MYBs were studied by the methods of RT-PCR,subcellular localization,transient overexpression,BSMV Virus silence,q RT-PCR,Yeast two-hybrid and GC-MS.The purposes of our study are to provide the theoretical basis for the molecular mechanism of Lilium ‘Siberia' fragrance formation by transcription factor regulation.The main findings are as follows:1.The cDNA full complete sequences of MYC2 transcription factors,named as LoMYC2 respectively,were isolated from Lilium ‘Siberia' flower by RT-PCR.The ORF c DNA of Lo MYC2 contained 2115 bp,and encoded 704 amino acids.Lo MYC2 is hydrophilic protein,the molecular weight is 76.6 k Da and the isoelectric point(p I)is 5.29.Lo MYC2 has a b HLH domain at the N-terminus and three HLH domains at the C-terminus,belonging to the MYC transcription factor family.The results showed that Lo MYC2 had the highest homology with MYC2 of Ananas comosus and Dendrobium officinale Kimura et Migo.2.The temporal and spatial expression of Lo MYC2 was analyzed by RT-qPCR.The results showed that Lo MYC2 was expressed in sepals,petals,stamens,pistils,leaves,stems,bulbs and roots,and the highest expression in pistils,followed by expression in petals and stems,and the lowest expression in roots.Lo MYC2 expression is not fully synchronized with the flowering process,with the coming out of flowers,the expression level showed an increasing trend in the buds and the initial opening stage,and dropped to the lowest value in the semi-open period,finally soared the maximum In the bloom and senescence period.3.The subcellular localization of Lo MYC2 was predicted,and the nuclear localization signal was PRPRKRGRKP at 501-511,suggesting that it was located in the nucleus.To test where is the subcellular localization of Lo MYC2,we fused full-length c DNA of Lo MYC2 with Green fluorescent protein(GFP),then introduced into onion epidermal cells,under 16 hours light/ 8 hours dark for two days.The empty control p35S-GFP was negative control,the nuclear green speckles were found from in onion epidermal cells of GFP: Lo MYC2,and at same time,the nuclear under DAPI light showed blue speckles,indicating that Lo MYC2 located in nucleus.4.To explore the role of Lo MYC2 in lily volatile scent,The BSMV-VIGS vectors of Lo MYC2 were constructed.It was used to transformed into Lilium petals by Agrobacterium.Then q RT-PCR and GC-MS are utilized to detect the change of relative genes expression and fragrance esmission amount.The results reveal that the Lo MYC2 gene expression fell down to 42.67% coupling with MYB1,MYB2,MYB3,AAT and TPS gene expression descreased about 47.33%,46.04%,71.08%,35.15% and 19.38%respectively.The value of the fragrance ocimene,linalool,methyl benzoate descended57%,79.36% and 73.15%.5.The vector of pOX: LoMYC2 was transformed into the petals by Agrobacterium.After overexpression of Lo MYC2,the volatile scent rosed respectively,but some relative genes expression dropped and some rosed.The expression of Lo MYC2 was soaring 89.27%,the gens Lo AAT and Lo TPS of biosynthesis volatile scent increased about 402% and62.82%,the expression of Lo MYB1 and Lo MYB2 that relative volatile scent transcription factor rosed 119.76%and1.2%,however,the Lo MYB3 dropped 37.16% respectively.Ocimene,linalool,methyl benzoate were soared of 1.8 fold,1.7889.3% fold,and 1.1 fold.6.Two bait vectors of Lo MYB1,Lo MYB2,Lo MYB3(full-length)and one prey vectors full-length of Lo MYC2 were constructed.The results of the yeast two-hybrid showed that Lo MYC2 can not only interact with Lo MYB1 but also can interact with Lo MYB3 respectively.