| There are many varieties of modern rose,which are widely distributed around the world.In addition to flower shapes and colors,the molecular mechanism of floral fragrance is also one of the important directions of rose research.According to the synthesis pathway,floral volatiles in plants can be divided into four classes: terpenoids,benzenoids/phenylpropanoids,aliphatic compounds and branched chain amino acid derivatives.Terpenoid synthase(TPS)is the key enzyme in the final synthesis of terpenoids.In this study,the TPS gene family was identified from the genome of Rosa hybrida,and a series of bioinformatic analyses were conducted.The differences of floral volatiles’ release amounts and composition in 9 different cultivars were analyzed by headspace solid phase microextraction gas chromatography-mass spectrometry(SPMEGC-MS),and eight TPS transcripts were cloned from the rose petals in the period of full bloom,of which the expression pattern and gene function were further analyzed.The main results of this study were as shown below:1.A total of 58 TPS genes were identified from the genome of Rosa hybrida.Phylogenetic analysis showed that the TPS family of Rosa hybrida included six subfamilies: TPS-a,TPS-b,TPS-c,TPS-e、TPS-f and TPS-g,among which the TPS-a subfamily was the largest,containing 39 genes.Members of the same subfamily shared similar gene structures and conserved motifs,and three conserved domains,DDxx D,NSE/DTE and Rx R,were ubiquitous in TPS proteins.The proteins encoded by the TPS gene of Rosa hybrida were all hydrophilic proteins,and most of them were unstable.TPS genes of Rosa hybrida were mainly distributed in tandem repeats on chromosomes.In the promoter sequence of Rh TPS gene,there contained a large number of cis-acting elements related to plant growth and development,stress response,hormone response and light response.2.A total of 79 floral volatiles were identified by SPME-GC-MS in 9 modern rose cultivars,including 29 aliphatic compounds,9 benzenoids/phenylpropanoids,and 41 terpenoids.It was found that the release amounts and composition of floral volatiles were significantly different among the 9 cultivars.3.A total of 8 transcripts were cloned from the rose petals in the period of full bloom,which were named Rh LNS1 a,Rh LNS1 b,Rh LNS2,Rh EBOS1,Rh EBOS2,Rh EAFS,Rh LINSa and Rh LINSb,and the first 3 transcripts belonged to the TPS-g subfamily and the last five transcripts belonged to the TPS-b subfamily.There is a special pathway for the synthesis of monoterpene geraniol and its derivatives catalyzed by Nudix hydrolase gene Rh NUDX1 in rose.Real-time fluorescence quantitative q RT-PCR was used to analyse the expression pattern of these 8 transcripts and Rh NUDX1 in the rose petals.4.Overexpression vectors and prokaryotic expression vectors of these 8 transcripts were constructed.The recombinant proteins of 6 transcripts except Rh LNS1 a and Rh LINSb were obtained by E.coli prokaryotic induction.FPP were used as substrates for enzyme activity assay of recombinant protein in vitro,and the reaction products were detected by SPME-GC-MS.The results showed that only Rh LNS2 and Rh EAFS could catalyze the synthesis of farnesene with FPP.The constructed overexpression vector was transformed into Agrobacterium GV3101,and then infected the somatic embryos of Rosa rugosa ‘Baobai’.After co-culture and resistance screening,the transgenic somatic embryos of 8 transcripts were obtained,which were preliminarily confirmed by fluorescence stereomicroscopy.In conclusion,the function of TPS family was preliminarily studied in this study through identification of TPS family members,bioinformatics analysis,floral volatiles analysis,gene cloning,real-time fluorescence quantitative analysis,prokaryotic expression and overexpression... |
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