Molecular Mechanism Of OjTPS1 Regulating The Biosynthesis Of Characteristic Flavor β-caryophyllene In Oenanthe Javanica(Blume)DC.

Water dropwort[Oenanthe javanica(Bl.)DC.]is a special aquatic vegetable crop belonging to the Apiaceae family.It is consumed as vegetable with unique flavor and rich nutrition.β-caryophyllene is the main substance that affects the fragrance and flavor of water dropwort,and makes great contribution to the composition of the flavor of water dropwort.It is identified as the characteristic flavor substance of water dropwort.However,there are few reports on the molecular mechanism of β-caryophyllene,the characteristic volatile flavor substance of water dropwort.In order to explore the regulation of OjTPS1 gene on the biosynthesis of β-caryophyllene and to provide a theoretical basis for futher analysis of the formation mechanism of volatile terpenes in water dropwort,the function of the candidate gene OjTPS1 involved in the biosynthesis of β-caryophyllene was verified.1.Determination and analysis of β-caryophyllene in water dropwortThe temperature rise procedure,shitter ratio and internal standard of SPME-GC-MS for the volatile substance determination of ’Fuqin No.1’were optimized.The optimized GC-MS conditions are as follows:initial temperature 50℃,15℃/min to 95℃ for 2 min,3℃/min to 100℃ for 1 min,3℃/minto 125℃,15℃/min to 155℃ for 2 min,and then 5℃/min to 185℃for 1 min.The mass scanning range was adjusted to 33-500 amu.The volatile compounds from the roots,stems,leaf blades,petioles and flowers of ’Fuqin No.1’ were determined and analyzed.A total of 17 volatile terpenoids were identified,including 9 monoterpenoids and 8 sesquiterpenoids.The accumulation of volatile terpenoids in water dropwort showed tissue differences.The highest content of volatile terpenes was found in flowers(1115.32 μg/g),which was significantly higher than that in other tissues,while the content in roots was the lowest(27.97 μg/g).The content of volatile substances in flowers was about 40 times that in roots.And the content of volatile substances from high to low were flowers>leaf blades>petioles>stems>roots.The content of β-caryophyllene in different tissues also showed tissue differences.The content of β-caryophyllene in flowers was significantly higher than that in other tissues,which was 131.34 μg/g.The content of βcaryophyllene in roots was the lowest(4.19 μg/g).The β-caryophyllene content in flowers was about 30 times that in roots.The content of β-caryophyllene from high to low was flowers>petioles>leaf blades>stems>root.2.Cloning and expression analysis of the β-caryophylene synthase gene OjTPS1The OjTPS1 gene encoding β-caryophyllene synthase(Genebank Accession No.OP593324)was isolated and cloned from water dropwort.The open reading frame length was 1692 bp,containing 7 exons and 6 introns.Phylogenetic analysis showed that OjTPS1 belonged to the TPS-a subfamily.The characteristic domain of OjTPS1 protein is highly conserved and contains several typical motifs of the TPS family.Such as the double arginine motif’RR(x)8W’ and the aspartic acid motif’DDxxD’.The expression of OjTPS1 gene showed tissue differences,and the relative expression of OjTPS1 gene in flowers was about 14.84 times that in roots,and about 3.22 times that in leaves.The expression leve1 of OjTPS1 gene was positively correlated with the content of total volatile substances and β-caryophyllene in different tissues of water dropwort,and the correlation coefficients were 0.826 and 0.928,respectively.3.Functional analysis of β-caryophyllene synthase gene OjTPS1Subcellular localization assay confirmed that OjTPS1 protein was localized in the cytoplasm.The recombinant OjTPS1 can catalyze the reaction substrate FPP to produce βcaryophyllene in vitro.OjTPS1 was overexpressed in Arabidopsis,and two transgenic lines(OE-1 and OE-2)of Arabidopsis overexpressing OjTPS1 were successfully screened.The content of β-caryophyllene in wild Arabidopsis and transgenic Arabidopsis were determined by SPME-GC-MS.The content of β-caryophyllene in two transgenic lines was significantly higher than that of wild Arabidopsis.OjTPS1 gene was silenced by CMV VIGS system.The expression of OjTPS1 gene was detected by qRT-PCR after silencing.The expression of OjTPS1 gene in CMV-OjTPS1 was 12.55 times lower than that in the control.The content ofβ-caryophyllene in CMV-OjTPS1 was 210 μg/g,and that in the control was 375 μg/g.After silencing OjTPS1 gene,the content of β-caryophyllene in leaves of water dropwort was significantly lower than that of control.